Method of Detecting and Treating Tuberous Sclerosis Complex Associated Disorders

ABSTRACT

Disclosed are methods of detecting and treating tuberous sclerosis complex associated disorders. Also disclosed are methods of identifying agents for treating tuberous sclerosis complex associated disorders.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/462,300, filed May2, 2012, which is a continuation of U.S. Ser. No. 12/080,362, filed Apr.2, 2008, now abandoned, which is a continuation of U.S. Ser. No.10/991,173, filed Nov. 16, 2004, now abandoned, which is a continuationof U.S. Ser. No. 10/016,253, filed Dec. 10, 2001, now abandoned, whichclaims priority to U.S. Provisional Patent Application No. 60/254,268,filed Dec. 8, 2000, each of which is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The invention relates to methods of detecting and treating TuberousSclerosis Complex (TSC) associated disorders.

INCORPORATION—BY-REFERENCE Sequence Listing

The instant application contains a sequence listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Sep. 29, 2014, is named21402-042C02_SeqList_ST25, and is 35,081 bytes in size.

BACKGROUND OF THE INVENTION

The phakomatoses, or ‘neuro-cutaneous disorders’, are a group of threeMendelian autosomal dominantly inherited diseases that present withphenotypes affecting multiple organ systems in affected individuals.Neuro-cutaneous disorders include for example, Neurofibromatosis (NF),Tuberous Sclerosis (TSC) and Von Hippel-Lindau (VHL). These diseases allproduce both neurological and dermatological symptoms.

Tuberous sclerosis complex (TSC) is an autosomal dominanttumor-suppressor gene syndrome, characterized by development ofdistinctive benign tumors (hamartomas) and malformations (hamartias) inmultiple organ systems. The brain, skin, heart, and kidneys are commonlyaffected. TSC lesions occurring in the skin and kidney contain smoothmuscle cells, endothelial cells, adipocytes, and large neuronalappearing cells. Despite this complex cellular architecture, kidney andother lesions in TSC appear to be clonal in nature, based on clonalityand loss of heterozygosity (LOH) analyses. In the brain, TSC producesboth subependymal tubers that line the ventricular sacs and subcorticalhamartomas which serve as foci for epileptic discharges. TSC producescardiac rhabdomyomas in the fetus/newborn that spontaneously regress inthe first year of life. TSC is also associated with renalangiomyolipomas, pulmonary symptoms, and manifestations in other organsystems. In addition, TSC is also associated with multipledermatological features such as hypomclanotic macules, facialangiofibroma, shagreen patches, and ungual fibromas.

A better understanding of the molecular nature of this disease willprovide new therapeutic tools to treat the pathologies associated withTSC complex not only in TSC patients but also in non TSC patientsafflicted by similar pathologies.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery of changes inexpression patterns of multiple nucleic acid sequences in cells derivedfrom the Tsc2 knockout transgenic mice compared to the expressionpattern found in cells derived from Tsc2+/− heterozygote and wild typesibling mice. These differentially expressed nucleic acids includepreviously undescribed sequences and nucleic acids sequences that, whilepreviously described, have not heretofore been identified as TSCmodulated.

In various aspects, the invention includes methods of diagnosing ordetermining susceptibility to Tuberous Sclerosis Complex (TSC)associated disorder, and methods of treating those disorders. Forexample, in one aspect, the invention provides a method of diagnosingdetermining susceptibility to a tuberous sclerosis complex associateddisorder by providing a test cell population that includes one or morecells capable of expressing one or more TSC modulated nucleic acidssequences. Levels of expression of one or more sequences, termed TSCXsequences, are then compared to the levels of expression of thecorresponding nucleic acids in a reference cell population. Thereference cell population contains cells whose tuberous sclerosiscomplex associated disorder status is known, i.e., the reference cellsare known to have or are known not to have a tuberous sclerosisassociated disorder.

The invention in another aspect includes a method of identifying atherapeutic agent for treating a tuberous sclerosis complex associateddisorder. The method includes providing from the subject a test cellpopulation comprising a cell capable of expressing one or more TSCXnucleic acids sequences, contacting the test cell population with thetherapeutic agent, and comparing the expression of the nucleic acidssequences in the test cell population to the expression of the nucleicacids sequences in a reference cell population.

The invention in a further aspect includes a method of selecting anindividualized therapeutic agent appropriate for a particular subject.The method includes providing from the subject a test cell populationcomprising a cell capable of expressing one or more TSCX nucleic acidssequences, contacting the test cell population with the therapeuticagent, and comparing the expression of the nucleic acids sequences inthe test cell population to the expression of the nucleic acidssequences in a reference cell population.

Also provided are novel nucleic acids, as well as their encodedpolypeptides, which are tuberous sclerosis complex modulated.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION

The present invention is based in part on the discovery of changes inexpression patterns of multiple nucleic acid sequences in cells derivedfrom the Tsc2 knockout transgenic mice compared to the expressionpattern found in cells derived from Tsc2+/− heterozygote and wild typesibling mice.

The change is expression pattern was identified by GeneCalling™ analysis(U.S. Pat. No. 5,871,697; Shimkets et al., 1999 Nature Biotechnology17:198-803, incorporated herein by reference in their entireties) ofneuronal stem cell (NSC) and mouse emroyonic fibroblasts (MEF) celllines established from 10-11 day embryos from mice of the threegenotypes (i.e.,)

A summary of the sequences analyzed are presented in Table 1. The 142single nucleic acid sequences identified herein, are referred to hereinas TSC 1-142 or TSCX nucleic acids or polypeptided. Differentialexpression of TSC 1-142 gene fragments was confirmed using a unlabeledoligonucleotide competition assay as described in Shimkets et al.,Nature Biotechnology 17:198-803.

By comparing the genes differentially expressed in both cell lines itwas possible to identify understand common mechanisms in TSC−/− tumorformation. Whereas, by comparing the genes differentially expressed inNSC cell lines it was identify genes that are expressed in cells thatare the originators (i.e., progentitors) of TSC tumors. Based on the TSCphenotype, genes that are up-regulated in the TSC-cells may have a rolein cancer progression, specifically for renal and lung carcinomas

Twenty-six sequences (TSC: 1-26) represent novel murine genes for whichthe sequence identity to sequences found in public databases suggestinga putative homology.

The 116 other sequenced identified have been previously described. Forsome of the novel sequences (i.e., TSC: 1-26), a cloned sequence isprovided along with one or more additional sequence fragments (e.g.,ESTs or contigs) which contain sequences substantially identical to, thecloned sequence. Also provided is a consensus sequences which includes acomposite sequence assembled from the cloned and additional fragments.For a given TSC sequence, its expression can be measured using any ofthe associated nucleic acid sequences may be used in the methodsdescribed herein. For previously described sequences database accessionnumbers are provided. This information allows for one of ordinary skillin the art to deduce information necessary for detecting and measuringexpression of the TSC nucleic acid sequences.

A subset of the TSC modulated genes can be further subdivided into threeclasses:

A. Secreted and/or Membrane Bound Proteins that are Up-Regulated in CellDerived from Tsc2 Knockout Transgenic Mice

Proteins in this category include, Plasma phospholipid transfer protein,Lysyl hydroxylase isoform 2, DVS27-related protein [AB024518], CathepsinL, Tenascin, ADAMTS1, Tissue inhibitor of metalloproteinase-2, Integrinbeta-5, Thrombospondin 2 (THBS2) Aspartyl protease 1, Cyr61, TetraspanNET-7, Cysteine-rich glycoprotein SPARC, neuronal pentraxin receptor,ITM2B-E25B protein Integral Membrane Protein 2B, transmembraneglycoprotein NMB, and zinc finger protein

These proteins are potential candidates for antibody screening andantibody-binding therapy for the treatment of TSC and TSC relateddiseases.

B. Secreted and/or Membrane Bound Proteins that are Down-Regulated inCell Derived from Tsc2 Knockout Transgenic Mice

Proteins in this category include, Growth/differentiation factor 1(GDF-1), Extracellular matrix associated protein (Sc1), Membrane-type 2matrix metalloproteinase and Thrombospondin 1 mice.

These proteins that are potentail canidates for the treatment of TSC andTSC related diseases.

C. Protein with Enzymatic Activities

Proteins in this category include Growth factor-inducible immediateearly gene 3CH134/erp, Galactokinase 1, Serum inducible kinase (SNK),PAF acetylhydrolase Aspartyl protease 1, Lysyl hydroxylase iso form 2Peroxisomal D2, and D4-dienoyl-CoA reductase (Pdcr).

These proteins are potential candidates for small molecule screening andsmall molecule drug therapy for the treatment of TSC and TSC relateddiseases.

The TSC modulated nucleic acids discussed herein include the following:

TABLE 1 MEF +/− TSC2 MEF −/− TSC2 NSC +/− TSC2 NSC −/− TSC2 TSCX SEQ IDvs. +/+ TSC2 vs. +/+ TSC2 vs. +/+ TSC2 vs. +/+ TSC2 Gene DiscoveredAssignment NO Acc # (16606) (16607) (16608) (16609) Novel gene fragment,2520 bp 1 1 aa914498 ±1.0 +1.5 +2 +1.5 Novel gene fragment, 1863 bp 2 2aa073509 ±1.0 −6 −2 −2 Novel gene fragment, 750 bp 3 3 AA183535 ±1.0 +3±1.0 +4 Novel gene fragment, 281 bp, 91% AA 4 4 ±1.0 −1.5 ±1.0 NEWidentity to rat Steroid sensitivity gene-1 protein [AAF35351] Novel genefragment, 1568 bp, 86% SI to 5 5 ±1.0 X +2 +6 human Tetraspan NET-7[AF120266]/old brain study also Novel gene fragment, 300 bp, 94% SI torat 6 6 O O O +15 10-formyltetrahydrofolate dehydrogenase [M59861] Novelgene fragment, 965 bp, 86% SI to rat 7 7 −2 X ±1.0 NEW myr3 myosin Iheavy chain [X74815] Novel gene fragment, 408 bp, 97% SI to rat 8 8 O O±1.0 OFF Limbic system-associated membrane protein [U31554] Novel genefragment, 777 bp, 83% SI to rat 9 ±1.0 ±1.0 ±1.0 NEW neuronal pentraxinreceptor [AF005099] Novel gene fragment, 354 bp, 87% SI to 10 9 ±1.0 X−2 −5 human KIAA0631 [AB014531] Novel gene fragment, 955 bp 11 10 ±1.0 X−3 −8 Novel gene fragment, 1113 bp 12 11 +2 X ±1.0 −9 Novel genefragment, 918 bp 13 ±1.0 ±1.0 ±1.0 +3 Novel gene fragment, 1166 bp 14±1.0 ±1.0 ±1.0 +10 Novel gene fragment, 594 bp 15 12 ±1.0 ±1.0 ±1.0 −10Novel gene fragment, 713 bp 16 13 O O ±1.0 OFF Novel gene fragment, 306bp, 95% SI to rat 17 14 ±1.0 −2 ±1.0 X ribosomal protein L13a [X68282]Novel gene fragment, 66 bp, 96% SI to rat 18 15 ±1.0 −2 −2 ±1.0ribosomal protein S20 [X51537] Novel gene fragment, 1613 bp 19 16 ±1.0+3 ±1.0 −5 Novel gene fragment, 2245 bp 20 17 ±1.0 NEW −2 −3 Novel genefragment, 171 bp, 86% SI to rat 21 18 ±1.0 +1.5 nonmuscle caldesmon[U18419] Novel gene fragment, 491 bp, 72 % SI to 22 19 +10 humanDVS27-related protein [AB024518] Novel gene fragment, 659 bp, 72% SI to23 20 −2 X ±1.0 NEW human ATP cassette binding transporter 1 [AF165281]Novel gene fragment, 341 bp, 84% SI to 24 21 human sorting nexin 5(SNX5) [AF121855], Novel gene fragment, 53 bp, 84% SI to rat 25 22calcium-independent alpha-latrotoxin receptor [U72487] Novel genefragment, 52 bp, 98% SI to rat 26 −2 Na+, K+-ATPase alpha(+) isoformcatalytic subunit [M14512] MEF & NSC −/− conserved differentialexpression Ribosomal protein L8 (RPL8) 27 U67771 −9 OFF −3 OFF Alpha-Bcrystallin (p23) 28 M63170 ±1.0 +20 +2 +7 Tumor cell dnaJ-like protein 129 L16953 ±1.0 +2 +3 +2 Insulin-like growth factor-binding protein-4 30S80566 ±1.0 −2 +3 OFF Insulin-like growth factor binding protein 5 31L12447 ±1.0 NEW +2 +5 (IGFBP5) Rac1 32 X57277 −2 −1.5 −2 −2 Growthfactor-inducible immediate early 33 S64851 ±1.0 +2 ±1.0 +6 gene3CH134/erp Phosphatidic acid phosphatase type 2c 34 AF123611 ±1.0 −5±1.0 −4 (Ppap2c) Annexin III 35 AJ001633 ±1.0 NEW ±1.0 NEWTaipoxin-associated calcium binding protein 36 AF049125 ±1.0 −2 ±1.0 OFF49 C-fos oncogene 37 V00727 +2 +1.5 ±1.0 NEW Stra13 38 AF010305 +2 +6±1.0 +2 E1B 19K/Bcl-2-binding protein homolog 39 AF041054 +2 +5 ±1.0 +3(Nip3) Peroxisomal D2,D4-dienoyl-CoA reductase 40 AF155575 +7 NEW +2 NEW(Pdcr) Galactokinase 1 41 AB027012 ±1.0 +4 ±1.0 +1.5 Alpha-enolase(2-phospho-D-glycerate 42 X52379 +3 +5 +3 +15 hydrolase) (NNE)Alpha-N-acetylglucosaminidase 43 AF003255 ±1.0 +2 ±1.0 +3 Uncouplingprotein 2 (UCP2) 44 AF111998 ±1.0 NEW +2 NEW ANC1 for adenine nucleotidecarrier 45 X74510 ±1.0 −1.5 ±1.0 −2 Vacuolar ATPase subunit A gene 46U13837 ±1.0 +3 ±1.0 +2 S-adenosylmethionine decarboxylase 47 D12780 ±1.0+2 ±1.0 +5 Spermidine/spermine N1-acetyltransferase 48 L10244 ±1.0 +5±1.0 +4 (SSAT) Xanthine dehydrogenase 49 X62932 ±1.0 +9 ±1.0 NEW MBOCT50 AB012808 ±1.0 OFF ±1.0 −3 Plasma phospholipid transfer protein 51U37226 ±1.0 +2 −2 +5 Lysyl hydroxylase isoform 2 52 AF080572 +3 +6 ±1.0NEW Cathepsin L 53 J02583 ±1.0 +5 ±1.0 +4 Ezrin 54 X60671 +2 +4 ±1.0 +4Thy-1.2 glycoprotein 55 M12379 −2 −4 ±1.0 −10 A-X actin 56 J04181 +5 NEW+6 NEW MHC class I heavy chain precursor 57 U47325 +3 +2 ±1.0 +4(H-2D(b)) MHC class I heavy chain precursor 58 U47328 +2 NEW ±1.0 +3(H-2K(b)) MHC region containing the Q region of 59 AP111103 ±1.0 +4 −2NEW class I NGF-inducible protein TIS21 (aka BTG2) 60 M64292 +2 +2 ±1.0NEW Ndr1 61 U60593 +2 +8 ±1.0 NEW Gly96 62 X67644 +2 +3 ±1.0 +2 p8protein 63 AF131196 +2 +4 ±1.0 +5 MEF & NSC −/− opposite differentialexpression Adrenomedullin precursor 64 U77630 ±1.0 OFF ±1.0 NEWFibroblast growth factor 65 M65053 ±1.0 −3 −2 +2 Serum inducible kinase(SNK) 66 M96163 ±1.0 −3 +2 NEW Annexin VI 67 X13460 ±1.0 −2 ±1.0 NEWAnnexin I 68 X07486 −2 −1.5 +2 +10 Annexin II 69 D10024 ±1.0 −4 +2 +2AP-2 transcription factor 70 X57012 ±1.0 OFF ±1.0 +20 Jun-B 71 J03236 +2−4 ±1.0 NEW PAF acetylhydrolase 72 U34277 ±1.0 OFF ±1.0 +12Phosphomannomutase 73 AF007267 ±1.0 +3 −2 −3 Sodium/potassium ATPasebeta subunit 74 X61433 +3 +8 −2 −12 Thioredoxin 75 X77585 ±1.0 +1.5 +2−3 Spermidine synthase 76 L19311 ±1.0 +2 ±1.0 −2 Aldehyde dehydrogenaseII 77 M74570 +2 NEW ±1.0 OFF Voltage dependent anion channel 2 78 U30838+2 +2 ±1.0 −2 Tenascin 79 D90343 ±1.0 −5 +2 +4 ADAMTS1 80 D67076 −2 −2±1.0 +2 Tissue inhibitor of metalloproteinase-2 81 M93954 ±1.0 −2 ±1.0+3 Integrin beta-5 82 AF022110 ±1.0 −3 ±1.0 +1.5 Thrombospondin 2(THBS2) 83 L07803 ±1.0 −6 ±1.0 NEW Membrane glycoprotein M6 = major CNS84 S65735 +2 NEW ±1.0 −4 myelin protein PLP/DM20 homolog Gelsolin 85J04953 ±1.0 −2 ±1.0 NEW Gag = antigen LEC-A, env 86 S74315 ±1.0 −2 ±1.0+5 NSC only 87 Quaking type I (QKI) 88 U44940 ±1.0 ±1.0 ±1.0 −1.5 mSin3B89 L38622 ±1.0 ±1.0 ±1.0 +2 Retinoblastoma susceptibility protein (pp10590 M26391 ±1.0 ±1.0 ±1.0 +1.5 Rb) Heat shock protein (hsp-E7I) 91 L40406±1.0 ±1.0 ±1.0 +2 Aspartyl protease 1 92 AF216310 ±1.0 ±1.0 ±1.0 +10Placental growth factor-1 (p1GF) 93 X80171 ±1.0 ±1.0 ±1.0 +3Growth/differentiation factor 1 (GDF-1) 94 M62301 ±1.0 X ±1.0 OFFCalgizzarin/ S100 A11 95 U41341 ±1.0 ±1.0 +2 +15 Cyr61 96 M32490 +2 ±1.0±1.0 +25 ADP-ribosylation factor-directed GTPase 97 AF075462 ±1.0 X −2−2 activating protein isoform b (Shag1) Camk-2 mRNA for Ca2+/calmodulin98 X63615 ±1.0 ±1.0 ±1.0 −10 dependent protein kinase MAPKAPK5mitogen-activated protein 99 AF039840 −2 ±1.0 −2 −2 (kinase-activatedprotein kinase Fyn proto-oncogene encoding p59fyn 100 M27266 ±1.0 ±1.0±1.0 −2 Beta 1,4N-acetylgalactosaminyltransferase 101 L25885 ±1.0 X ±1.0+1.5 Muscle glycogen phosphorylase (Pygm) 102 AF124787 ±1.0 X +2 OFFProtein phosphatase 1 binding protein PTG 103 U89924 ±1.0 X ±1.0 −4Argininosuccinate synthetase (Ass) 104 M31690 +2 X ±1.0 NEW Phospholipidhydroperoxide glutathione 105 AF045769 ±1.0 ±1.0 ±1.0 +5 peroxidase(Gpx4) GABA transporter (GAT4) 106 L04662 ±1.0 ±1.0 −2 OFF Sodiumbicarbonate cotransporter NBC1 107 AF141934 −3 X −3 −2 Glial fibrillaryacidic protein (GFAP) 108 K0I347 O O O NEW Tropomodulin 109 S76831 ±1.0X ±1.0 NEW Cysteine-rich glycoprotein SPARC 110 X04017 ±1.0 ±1.0 −2 +10DSD-1-proteoglycan 111 AJ133130 ±1.0 ±1.0 ±1.0 OFF Extracellular matrixassociated protein (Sc1) 112 U64827 ±1.0 X ±1.0 −1.5 Membrane-type 2matrix metalloproteinase 113 D86332 O O −2 −8 Astrotactin 114 U48797 O O−2 −10 Adipose differentiation related protein 115 M93275 ±1.0 X +2 NEW(ADRP) Ventral neuron-specific protein 1 NOVA1 116 AF232828 O ±1.0 ±1.0OFF Neuronal pentraxin 1 (NPTX1) 117 U62021 −2 X ±1.0 −10 Receptoractivity modifying protein 1 118 AF209904 ±1.0 ±1.0 +2 −7 (Ramp1)Lunatic fringe 119 AF015768 ±1.0 X ±1.0 −4 TPA-induced T1S11 120 X14678+2 ±1.0 ±1.0 +6 ITM2B - E25B protein Integral Membrane 121 U76253 O O +6NEW Protein 2B NMB 122 aj251685 −4 X ±1.0 NEW B-cell translocationgene-1 protein (BTG1) 123 L16846 ±1.0 ±1.0 ±1.0 +2 MEF only Keratinocytegrowth factor/fibroblast growth 124 U58503 ±1.0 −10 +3 X factor-7 NOVprotein 125 Y09257 +2 OFF ±1.0 X TGF-beta binding protein-2 126 AF004874±1.0 −4 +2 X GATA-6 = zinc finger transcription factor 127 S82462 ±1.0+4 ±1.0 X PDGF-alpha-receptor (PDGF-alpba-R) 128 M84607 −2 −6 ±1.0 XVascular smooth muscle alpha-actin 129 X13297 ±1.0 −6 ±1.0 ±1.0 Alpha-2collagen VI 130 X65582 ±1.0 −8 ±1.0 X Laminin alpha 4 chain 131 U69176±1.0 −4 O O PGI (biglycan) 132 X53928 ±1.0 −5 +2 X Thrombospondin 1 133M87276 −2 −4 +2 X Fragile X mental retardation syndrome 134 L23971 ±1.0+3 ±1.0 X protein (Fmr1) Osf-2 for osteoblast specific factor 2 135D13664 ±1.0 −20 ±1.0 X Ndr2 136 AB033921 ±1.0 +10 +2 ±1.0 P53 137 X00741±1.0 Tuberin (Tsc2) 138 U37775 ±1.0 X −2 OFF Alpha glucosidase II alphasubunit 139 U92793 ±1.0 ±1.0 ±1.0 + DAN 140 D50263 ±1.0 Q ±1.0 +3intracisternal A-particle element 141 D49812 ±1.0 ±1.0 +2 +5 Annexin V142 U29396 +1.5 Key = New = de novo expression Bold = gene was confirmedin that job +1.0 = no difference X = no poison Q = in process p =partial poison O = no band

Below follows additional discussion of nucleic acid sequences whoseexpression is differentially regulated.

TSC1

TSC1 is a novel 2520 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 1) 1GGCTCTGGCTCGGGCTCGGGCTGGGGCTGGGGCTTGGGCTCCAGCTCGGGCCCTGCACCTGTGACTCGGCGGCGTTGCTC81CTCCGCTGCCCCATGGCCCCGTCCCGGCTGCAGCTCGGCCTCCGCGCCGCCTACTCCGGCTTCAGCTCGGTAGCCGGCTT161CTCCATCTTCTTCGTCTGGACGGTGGTCTACCGACAACCGGGGACTGCGGCGATGGGGGGTCTCGCAGGTGTCCTGGCAC241TGTGGGTCTTGGTGACTCACGTGATGTACATGCAGGATTACTGGAGGACCTGGCTCAGAGGGCTGCGCGGCTTCTTCTTC321GTGGGTGCTCTCTTCTCGGCAGTCTCCGTTTCCGCCTTCTGCACCTTCCTGGCATTGGCCATCACCCAGCATCAGAGTCT401CAAAGACCCGAACAGCTACTACCTCTCCTGTGTCTGGAGCTTCATTTCCTTCAAGTGGGCCTTCCTACTTAGCCTCTACG481CCCACCGCTACCGGGCTGACTTTGCGGACATCAGCATCCTTAGTGATTTCTAACCCAGGGAATGAGGTCACCACAGCCTG561GGGGCCCTCGGGATCTGGACTCAGCTTCCGAGTCAGCAAGGGAGCTCACCCCAACCCCTGGGGAACTCCAGAACCATGGC641AGAGTATATGGGCCCGTTCAGTTTCTCAGAAATCTGTCTGGTCCCCTTTTGGGGAAGATATAGAGCTGTTAAAGGGATAC721TGCCAATCTGCCCAATCTGCCCGTTAGCCCAGCTAGAGGGCAGCTTAGACCTTTCCAAATAGATCTATTTTCTTAGCCCT801CTGAGGGATCTCTGTAAGTAGGGCCACGACAATGAATTCAATGGGTAGGATTGGAACTATGGCTAGTGACAGGGGCTGGG881ACAGGCTTCCTTGCTACCCCAGACTTCATTGAAGCTGTGTGTGGGGGAGGCATCAAAGGTCTGGTCAAGAGAGGAATCTT961TAGTACAGATCTCCATCCCCTGTTCCCCACCCTGTTACCCTGAAGTGTCGGGTAGCCAAACTCACCGGTCCTTAGGGAAT1041TGACAATTGGCTCCTTCCCTAAGCAGCACAGTTGGACAGAATCCAGCGTCCGTCCGTCCTACCTTCCCATCCAGAGTTTG1121TTTCCCATGAGGGTGCTAGCGCCAGCCAACCATTCCCATGTGTCGCATATGCACACATGACCACACACACCAGAGCAGGA1201CTCCTCGGATGAGGCTAGACTTGAGGACCACAGGAAACACACCCCTGCACTTAGAAGGGCTTTGGGATCGGGGGCAACCT1281GGTGGGGGCAAGTGGGAGCTCTCCATCTGTACTGAGTCTCCAACCTTGCCCCTCACTGCACAAGACCACCCTGACCGTGA1361GGACCTCCTCCCTGCACCAGATCCTAACTCTGACCTTTCACCTTCTCTCTCTCCTGAAGGAACTCTTCTGAGTGGACATG1441GGCCCAAGGCCTTACCTAAGCGGAGAGGGAGGGCAGGGGCTGCTACTCTTCTCTGTAACCTTCTCTGATGGGTTGTCACT1521TTGCACGTCTACTCTTCCACTTGGGCACTGCCCCCAGCTCTCTGCCTTACCTGTGTTATGGGCACTTAAGCAGAAATACA1601GCGGCCATTTTAACCAGCAAAAAAAAAAAAAAATAGGGGGGTGGGCGGTTTTGAGAGGGGACAAGAGTGGGCAAGATGGG1681GGCTCTAGCTGTCTGATCATCTCCCTAAGTTTGGGGCTACTAGACGGTATTCCTCATCTCTGGTCCCCTATGGGAGACCA1761CCAGCTGAGATCTCCTTTGCTCTCCCAGTTCTGTCCCAGCCAGGGTTAGGATGCCCACAGACTCAACATCCCTGCAGATT1841CCATCTCCCCACCCTAAGCCAAGGTAGATGGGAAAGGGAATCTTTCTTTTTCTACCCCAGCCAGACTACTTGGGGCTCCA1921AGTTGACCAGGATGTGTGGATTCAGAAGCAGAAAGGCAGGAGCTAGCACCTCTCTCACGCTGGGTACACTTGTCCTGGCC2001TGTGTTTGCCTCACCCTGGCCTTTACAGTGTAAAAACACCATGGGACTTTAGAGCAGGGAAGGATAAGGAACAGTGTCAC2081TTCTAGAGCCTTCTGCTGGTAGACGCTCCTACTGATAGAGGAGGTAAAGACTACTGACCTCCCGGCTAGGCCTGGCTTAA2161GCCAGGCGTGGCCTGCGTCACAACCTTTTGCGGTGTCTTAGCAACCTGAACCTGAGATCTTATTCCCGAATCCCACAGGG2241CCCAATGTGCAGGGCTCAGCCTGGGGCCATCTCCCTTTTCACCTGGGTTGGTGAGCATGTATTTGGAGTGGTTTCTTCCT2321GCATGTATTAGCCAAGGAAGGACAAGGGACTAGAGGGTCTGAGTTAGGTCCAGACTTGTCCCCTTTCCCCAGCCCATCAC2401AGGATGCTGGGTGCACACCCACTCCACTGACGATGTCCCACCAACATCCAGGAGGCGTTCTCCCAAGGACTTTAAAGCAA2481 ATAAAACATATATTGTTCAGAAAAAAAAAAAAAAAAAAAA

TSC2

TSC2 is a novel 1863 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 2) 1AAGCGTGACCCTAAGTCTAGCCTGGAGCCAGGGCTAGAGTGGTCATTTCTTTGTGGGGTGCTGCCAGGGAGGGGCCAGAC81CCACAGGCTACTCAAAGGGCCTAGAGACCCCTCCCCAGGCAGGTGCTGCCCCAGGAGGAGCATGTCCTGGGGTCCGGGGA161CTGAAGTCCATGTGGCCTCAGCCCCCCACACCCAGAACACCGCTTGCCTAAGGTGCTTTTGGCTTTAGTGTGTGATGTTT241GCTGTGCTTCTGGGCTGAATTAGCTTCCAAATCAGGACCTGGAGCCTCTACCCTGGCCCAGCCAGCCAGTGTGAGCTCTG321GTCTGTGAGATGGGCAGCTACGGGCCAGTGGAGCAGCATGTGGTGGGAGGGGCAAGGCTGGGACCCAGTGGTTTACAGAC401CTGTGGCCCTCCTGGAGCAACCTGGCAGCTACGGATCCCAGAACCCCCTGGGCTTCAGCTCCCCCAGAGGGGAGAGGCTC481CACGTTGCTTTCCTTCCCCAAAATCCCTTTCTTTGTGCTGGTGTCTGGGACCAAAAGGAGTGGGCAGAGGACTCGGAGGG561CCTAGGGGTCCCAGTCGGGGCATCTGTAGCTCCTAAGCACGACAAGCATCAGTGCAGGGGACCCTGGCCTTGACTCCAAC641TGGCCTGGCGCCAGGAACCTCCAGGGCCAGAGCAGCCCAGCTGCAGCCAGCCTGCCCACTATGGGTATGTTCCTGGCCTA721AGGTCCGGAGGGAGGTTTGGGGTATCCCTGCCTGGGTGCCTGGGTGTGCCCTGGGGCCTCTCAGAAGCACAAATGCTGCC801CCCTGGCCGTGAGCAGGCCACAAGGTGAATGTATATAGCATGAGAGGCGGGCACTGCCCAGACGTGGCTGTGAACTTGTG881CTGTCTCGGGAGTCCTGACCTTCTGTGCGTGAGTGCCCCCATCTGTGACGTTTCACTCACCGAGGCTGAAGAAAGGAAGC961AGGGGAAATGAAAGCAGGGGTTTCTCGCCCTGACCCCTGCGGAGGAGACGGCTCCTACCACTGCGGTTGGCTTCATTTCG1041TTTTCCTGATTTCTGGGGTGCCACTTACCTACTCAATCCCAGTGGTCCACCCCCACATCCCCAGGGAGTGAGCAGTCCAG1121TGCCACCTGCCTGTGATTGGTCCCCAGTCCCTATTACCCAAGGGGACCCTACAGCTCTGGTGGGTAACAAGGAGGGCTAA1201GCCACCAAACCAGAGCCCGATCCCTTGCCGACCCAGGAGGAGGGATCTGGCTGAGAAAACTGATAGGACTGGAGGCCCCC1281ACCCCAACCAACACTCTCTGGTTTATGTGAGTAGCAGAAGATCCCGGCCTGGAGCATCCTTCAAGCCCTTCTCCCTGTGC1361CCACCCCGCCCCCCCCCCCCCCCATATCACTATGCAATTCTTGACCCCAGCTCCAAAGCTTGCCCTACCCGGTCCCAGCT1441CTGTCCGGCCCAGAAGGTGGCTAGCTGGTGGGCCACAGGTGACCAGGGTCTCTTTGTTTTTCATCACAGCGGTGGTGTGC1521CGCACCCTTCCTCCCATATGTGATTTTGTGAGATTGCCTCCCAGTTACGGTCCCTCTGCCTGCATCTGCCCCCAGTGGAC1601TATGTCATCTGAATCGAGCCAGCCCCAAGTTCCCCTCCAGCCTCTGTAGGGCCATGGCTGTGTGTTACTGTTGCTGTGCT1681TTCATTTTTTAAACTGGGTTTGGGGTTTGATTTTTATTTCTGTGGGGAACTTTATTTTTCTTGGCAAATAACTAAAGTTC1761TTGTCCATGTAATTTCTGTGGTCTCTATTCAGCTTGGGTTTCATGTTTTAAAATAAACAATTTTAAGAAACAAAAAAAAA1841 AAAAAAAAAAAAAAAAAAGC

TSC3

TSC3 is a novel 750 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 3)CTTGTTTATCCTACTCGGGTAGTTTCCTACTAATTTCAAGACTAGTGTTAACATTCTAAGGTAGTTATCTTAGGGTAGAT81TCAAGGTTTTAGATGACTAACAGTTCAGATTTTCTGATCAATTTTTTAAACACTAGAGAATAAAAGTGTACTAGAGAATA161AAAGCAGCTTCATAGTTAATTCTCACCAATTGGCCCTTTGCTAGCTGCTGGCTTTAGGTACACATAGGATAATATGTGTC241CACGTTTCTACTTGGAACTGGTAAAAGTTGTCACTGGCTGGAAAATGGTATCTCTCTCTTGTATACAAGATGGTCCATTG321ACACTGGTACTTTATGAAGCAGTTCTTTGTTTGTTTGATTGAGCTCTCTTGAACCTTGTTCATCTTTTAGTTTTTGCTTG401GAATGGAATGGAACTGGTTTGAAGTTAAAGGAAATATTCATTTTGAAACTTGTTCATTTTGAAAGGAAATGCAAGTTTCA481AAATGAAAAATAAAATGAAAAAGGAAATAAATTATTGTCCCAGATGGTCACTTGAGTTTTAAAAAATGGCTGCACACAGT561AAAACTGCTAAAAACAAAAACTTACCTCATTATTGGTTTGCATCTTTTTTCAGCTACTAATTTTATACCAAAATGTTAAA641TATTTATATTGTTTGAGTTTCAATCTTGTATGGAAAAAAATAATTAGTAGGTCTAAAAATGCCATGCTTTCCAATAAAGA721 AGTTAAAAAAATCATCAGTAATGTGAATTT

TSC4

TSC4 is a novel 281 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 4) 1GGGCCCCTCCGTCTCAGAGCAACTATACCCTCTACCTCGGAAGGAGCAGCAGAGAGAGAAGCCACAGGCCACCAGGAGGC81CCAGCAAAGCCACCAACTATGGAAGCTTCTCAGCCACCCCACCTCCCACCCTCTGGGAGGTCAGCACAAGAGTTGTGGGC161ACAAGCCGTTTCCGGGACAACCGGACAGACAAACGGGAACATGGCCATCAGGACCCAAATGTGGTGCCAGGTCCTCACAA241 GCCAGTAAAGGGGAAGCTGCCCAAAAAGAAGGACAGAATTC

TSC5

TSC5 is a novel 1568 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 5) 1CGCGCGGGAGCCAAGATGCCTCGCGGGGACTCGGAGCAGGTGCGCTACTGCGCGCGCTTCTCCTATCTTTGGCTCAAGTT81CTCTCTCATCATCTACTCCACCGTGTTCTGGCTGATTGGGGGCCTGGTCCTGTCAGTGGGGATCTACGCAGAGGCAGAGC161GGCAGAAATACAAAACCCTGGAAGAGTGCCTTCCTGGCCCCCGCCATCATCCTCATCCTCCTGGGGGTGGTCATGTTCAT241CGTCTCCTTCATCGGGGTGCTGGCTTCCCTCCGGGACAACCTGTGCCTTCTGCAGTCGTTTATGTATATCCTGGGGATCT321GCCTGGTCATGGAGCTTATTGGTGGGTCTGTATTTAGGGGCCGCCGGAACCAGACTATTGACTTTCTGAACGACAACATC401CGGAGAGGAATCGAGAATTACTACGATGATCTGGACTTCAAGAACATCATGGACTTTGTTCAGAAGAAGTTCAAGTGCTG481TGGCGGGGAGGACTACAGAGACTGGAGCAAAAACCAGTACCATGACTGCAGCGCCCCCGGGCCCCTGGCTGACGGGGTTC561CCTACACCTGCTGCATCAGGAACACGATGTTGTCAACACCATGTGTGGCTACAAAACAATCGACAAGGAGCGCCTGAATG641CACAGAACATCATTCACGTGCGGGGCTGCACCAACGCCGTGTTGATATGGTTCATGGACAACTATACCATCATGGCGGGC721CTTTTACTGGGCATCCTGCTTCCTCAGTTTCTTGGTGTGCTGCTGACCCTACTGTACATCACCCGTGTGGAGGACATTAT801CTTGGAGCACTCTGTCACGGATGGATTGCTGGGACCTGGTGCCAAGTCCAGAACGGACACAGCAGGCACTGGATGCTGCC881TGTGCTATCCCGATTAGCTATGCTGATTGAGCTATCCTGGCCCGGCACAGCAGCTCCCAGCCGGACTGTACTGCAAAGTG961CATCTAAGACTACACAAGCTGGACAGGACCAGCTGCAGCTCCTCTGCCCACCCACGGCGCTGACCAAAGCCCAGGGTGTA1041TGTACCTGCGTATAGTGTCTGATGGCCACTCCTCCTAGGGGAAAGCTGAACCCTGTGGGATCCCGGGAACAGGGATAGCC1121CAGCTCCGGTTCTGAGTCCTGGAGAAGGCAGCTCAGGGCTCCGTGTGGGCTCTTTTTCTTTCTGGCAGTGCCTTGGCCAG1201TGGTCATTATGCCCCTTCAAGGGCAGTTTTGCAGTGATTATTTTTAAAGGCAAGAAGGGAGTGTATCTGTTCTATAGGGA1281AGTCCTGGGTGCAGCCCTGGTACACTACTCTAGATGTGACGTTGGACTGTGTCTCAAATTCCCAGGTGCCTTGAGTCCTC1361TGTAAGGCTCCTGCTTTGCCCACCCATTTTCTACATATGTTTTTTTTCTTTTTTTTTTTTAATAACCGTGTTTTGTATAC1441AATTAACAAGAGTTTCTGGCTATTCAAAACTAGCCACCCCTGACCGAGTCCACTCACCCCTCCCCGTTAGTTCATTAATT1521 GAACAATAAATATGTGTTTTGGGGGGTGGTCTTTAAAAAAAAAAAAAA

TSC6

TSC6 is a novel 300 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 6) 1 gccggctctt tgtggaggac tccatccatg accagtttgt gcagaaagtggtggaggaag 61 tagggaagat gaaaatcggc gaccccctgg acagggatac caaccatggcccgcagaacc 121 atgaggccca cctgaggaag ctggtggagt attgccaacg tggtgtgaaggaaggggcca 181 cactggtctg tggtgggaac caagtcccaa ggccaggctt cttctttcagccaaccgttt 241 tcacagacgt ggaggaccac atgtacatcg ctaaggagga gtccttcgggcccatcatga

TSC7

TSC7 is a novel 965 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 7) 1CCCACAGCTCCTGCCCACTCACCAGGTCCAGGGGAGAGCAGGCGGTGACTCGATGACAAGTGCCTTTAGTTGAAGAGCAC81ATCTCACTCATTCCTCTCTCAGTACCTGATACATTCCTCTGTGCTAACCCCCCCTTGGGGAGGACCCACCCTCTGGAGGC161TGGACTTGGGGCGAACAGGCACTCACCTGTCACTGCCAAGGGCGGGCAGGCCATCCTTCCGAGCCCATGGGAGCCGGGAC241CACTAAGACTGCTGGTGGGAAGAAGTTGGGTGCTGGGCTGATGGTCTTGCTTTCTCTTGGTCTTCGCTTGTAATGTGGCT321GGCCCATGTTGGTTTTATGTTTAATGCTGTGCTTATAATAAGAAAGAGCCCCCCCAAGCTGTACATTTATAAAAAGTGAT401CATATACTGTATATAGAAAAATCTAGAAGCACATATGAATGCAGCAGGTAGTATTCCACTGTACCCATTCATGAAGGTAG481GTTTTATTACAGGACTCGCACCAGGTACTTACAGACGCGCCCTCTCCTCTTTGCCTAGAGAAACAGTCACTGCATTCCCG561CACAGTCCCTCAGACCCCCTTACCCTCTTCCCTGTAGGAAATTCTCCTGTGACCCCTCTGCCGTCCTCCCTTACTTCCTA641AATAAATGTAACGGAGTCAGTGCAAAAAAAAAAAAATAAATGACATTTATTGTGGGTTATAATTTTCTCCTAAAAACAAA721ACCAGTGGTATGGTCATACCCACCATTGTTTCCCCACTTTCCATGACCGTCACAAACATCTGGGATGAGCACCTTGTGAG801CAGGAAAAGTTATGCTTTAAGAAATTTCTGGCCAGGCGTGGTGGCATACACCTTTAATCCCAGCACTCGGGAGGCAGAGG881CAGGTGGATTTCTGAGTTCGAGGCCAGCCTGGTCTACAAAGTGAGTTCCAGGACAGCCAGGGCTACACAGAGAAACCCTG961 TCTCG

TSC8

TSC8 is a novel 408 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 8) 1 gccgggtctg aaaaggacta ggctggcatt ggtgacaccg agcttgttggcagccacaca 61 ggtatagttg ccatagtgtt cctcagtgac attggtcacc gtcaaggaggactggccctc 121 agtgctctta atctcaaggc catttgcact gtttatcctg gtgtcatcccggtaccactc 181 aaagtcaggt gcaggcaccg ctgaggcttc acatttgagg gaagcttgtcgtcctgtggt 241 ggcttcgttg ctcttcgact ccgtgatagt gggtggatag ttcacagtgaccttgacttg 301 tttgacatcc gccgaggaga cctcgttggc agccttgcac tcatatttgcctgactgttc 361 cctggtgatg cctaggatct ccagatattc ttcttctcct tcaaatty

TSC10

TSC10 is a novel 354 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 9) 1 gtgcaccaga tgttctacga ggccctagat aagtacggga acctcagtgctctgggcttc 61 aagcgcaagg acaagtggga gcgtatctct tactgccagt actacctgattgcacgcaaa 121 gtagccaaag gcttcttgaa gctcggccta gagcgtgccc acagcgtggcgatccttggc 181 ttcaactctc cagaatggtt cttctctgca gtgggcacag tgttcgcagggggcattgtc 241 actggcatct acaccaccag ctccccggag gcctgccagt acatctctcatgactgccga 301 gccaatgtca tcgtggttga cacacagaag cagctggaaa agatcctgaagatct

TSC11

TSC11 is a novel 955 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 10) 1CGGATCATCTGGGTCGCGACCTTGAGGCCGGGAATCGAGTTTCCAAACGTGCGGGGGCCTTCGCCGGCTCTGCTGCCCCC81TTTCTCTCCATGGCAGCGGCCCGGAACCTGCGCACCGCGTCATATTCGGAGGCTTCATCTCCATGGTCGGCGCCGCCTTC161TATCCCATCTACTTCCGGCCCCTTATGCGGCTGGAGGAATACCAGAAGGAGCAGGCTGTAAATCGAGCTGGTATTGTCCA241GGAAGATGTGCAACCGCCAGGTTGAAAGTGTGGTCTGATCCATTTGGCAGGAAATGAGGCTGTCAGCAAGTCTGATGAGG321AAAGTGGACGTCTTTATCCTGTGCACTCCGCAGTGGGGACAATAGATGCCTCACTGTGGCAGCATGGCATGGAGAGGGAA401CTCTCATGCTGCTAGCCAGACCCCTTGTGATAGAGACTGTGTGCAAAGACAGTGCTTCCCTTAACTCCCTGGAGAACCTG481AACAGATGCCACCATTAGGAAGTGCCTTGCGGCTCCATTGACTTTGCAGGAGCAGAGCCAGCCTGCAAGGCTGTTTGTGG561AAGATCTGCTGCTCCTGCAGTCTTTATCACTTCCAAGCTGTGATGTGAACACAAGCAACCTGTGGGCTCAAGGTCCGTGG641CTGCTCTGACACCTTTTGAATAAGCGATTTCAGTGCAAATGGCCTTGCCAAGCTGCCTCGCAGGGTTCTTGGAGGATGTT721TCAGTTGATAAAACTGTTTGAAGACAGGATCCTTGGCACTGTTTAAGAATATACACTGCTCAGCTTAACCATTTCATTGA801AAGTCACTGTGTGTGGAAGTGAATAGGGAGCGAGTCACACTAGACTATACCACACACAGTAGATTCCTGCGTGAGGCTGC881 AGGTATTAAAATGGTTTCTCTTAAAAAAAAAAAAAAAA

TSC12

TSC12 is a novel 1113 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 11) 1GGAGACCCAAGATCTGAACCAGCCAGCCAGGTGCTGCACAGCCTCAACTTTGGGAGCAGAGGCCCTGTGGGGTTAACTTG81GGTCTGCCAGAAACAGTGCTTCCCGCAGGGAAAATCTTGGGTCAAGATGGAGGCTGCTCTGGAACACTGAGTGTTTCAAG161GGAGAAAGAGTGGGAACCGTGGCCCTTTGGGGCCAGACCCTGCAGGAGCTTGCCTCGCCTTTGAGGAGGAGGCACTGCTC241TTCAGGTGCCCTGGAGGGGCTTTTAGTGCCATCCCCACAGCAGAGTAAAGGTGGCGCGTATGTCATCGGGTGGCTTTGCG321CTGGTAGAACGCTGTTCTCTACCCTGCTGCAGCCTTTCACACTCACACACACCCAAACACACACTTCTCGGCCCTGTATG401TTCAGGTGAGAGACAAGGGAAGATGGCTCATCATTTTCAGCCATGTCCCCAAAGTGGCCTCTCTTTCATGCTCTGTGGGC481TTTGGCCTGCAGCTGTTCCAGAGTTAGGGATGTGATTTTTGTCTGTGAGGTACCCCTTGCCCTAGTGGATCAGTTACAGG561CCTATGTCCAGCACCAGAGTCCCTGTTCCGATATCATCACAGATAGCCTGTTGTTTTCCACAGAGGAGCCAGATGTAAGT641CAGACACCTCCAGCCTACCAGTCTCCTGCCATCAGCTTTGGCTCTAATGGGCTCTTGGTGGCCTCCTTGGTGTGTCACTG721GTACAGGACAGCAAGTGGCTCAGAAAGGCTGCTTGCTCCTGAGCTCAGCCACTTATTCACATGGTTCAGAGCAGATCTTT801GTACTCTTCAGACTCAAGTATGGTGATCTGTTTGACAGTAGAGGTCTGGCCTACCCCTCACCCTCATTCTCCAGCACCTC881TAACAAGAACCACACTCATGCCTCTGGTGTCAGTTTTCTTGTCTGCCTTCCCTGGCCTACCTAGATATTTATTTCTTGTG961TTTTATGAATAGTTAAGCCCTGCCCATCTGTGCCTTTCAGACGGAAACACAGAAACCTAGGCTGTGCCATTTGTCTTCTC1041ACAGTTGTTTAATGAAACCTCAAGGAATATGGAAATAAAGCCTAGACCCTGGAGTGGTGAAAGAGTAAAAAAA

TSC15

TSC15 is a novel 594 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 12) 1AGATCTCTGTTTCCTCTTTCTTCTCTCCTCTATGCTCTTCTGTAGCCTACCCTCAGGGTGATCTCTAACCCAAACTAATC81CCGAGGAACAGACACTTGGCTCAGCTCCACCTACTACCTGGCTCACCTGTTCCCAGAATCTCCATAGAAGAGGGCACTTT161CTTTCTCAAGTTACCCTAACATTCTCTGCAGGATAAAATCATGAGTCCAGCCTGTCTGTGGAACTGGGGCCTGTCTGCAG241CTTCCCTGCAGAAGTGTCCATTCACTTTGGGTGATCTTCCCGACCAAGATACTTAGGTGTTTTGGCCAGCACCAGTATTT321CTATGAATTCCTGATCTGGAGTTGAATAGACAGGAATCAAGACCTAGGCTTTTCACTGTGTGAACCTGAGCATGTGGCCT401GACCTGCTGGAAGCTCCTCTGCTCTTGTGTGAAGCAGGAATGCTGTCAGGCACACAGCACAACACACCAGTGGTGGAGAA481CGCTAATCCCAACACACAAATTCCACAGAAATGGCACTATCCTCGGGTCTCCTGCCTAACCATGGACAAAGCTGAGAATA561 AACAGTGCTTTACTTTGAAAAAAAAAAAAAAAAA

TSC16

TSC16 is a novel 713 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 13) 1CAATTGTTTTTTCTAACCATCTTAGGGAACAATACATTGCAATAATTGATAATAGTGCCATCACTGTAATAAACTTTAGA81GACTTTTTTTAATGTAAAAGTTGTTGGTCACCTTGTTTCCTGTAACCTTCACTCTGTCACACGAGTTGGCTCATAGGTTG161TGTTTGTCTATCAGAAATAAGAAAAACACAAGTGAAGAAAATGTTGGCATGAAGTCATCCATCTGCAATGAAAAACCTAA241AAGACTACGGGTCACTCATGTTATCAATATAATTTATAATCCTGTTCAGTGTACAAAATTGTGGGTTTTGTACTCACCCA321AAAGACTAAAACACCAGTTTTTCTTACAGTATCTATCTACAGAGCTTATTCTCCCCTATTATTTGGGAAACTCTGAGACT401CCATATTGCAGAAGTCAAGGAATAGGCCATATAAGAAAATGTAGCTTGTTTTTATTATTTCTGCATATTTATTTCTAGAT481CTTGGGCTCATTTGTTAACAGAATAAGTTGTCAAAGGTAAAGTCCTTGAGTCTGGGAATGAGCCATCGTTCCAAAACCAA561CACACCCTGTGTGGAAATTTTACTTGACTCTGTTTTGCTGCATAGAATTCAGTGTCTCTTGGCCATTCCCCCTCATTCCT641ATACTAAATTCTTTGAAGACACTGGTAACAGTTTGTGGTAGACTACAGTTGAAAAAACTCAATCCTTATTTCT

TSC17

TSC17 is a novel 306 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 14) 1 ggatccctcc accctatgac aagaaaaagc ggatggtggt ccctgctgctctcaagggtt 61 gttcgcgctg aagcctacca gaaagtttgc ttacctgggg cgtctggcgcatgaggtcgg 121 gtggaagtac caggcagtga cagccactct ggaggagaaa cggaaggaaaaggccaagat 181 gcactatcgg aagaagaagc agatcttgag gttacggaaa caggcagaaaagaatgtgga 241 gaagaaaatc tgcaagttca cagaggtcct caagaccaac ggactcctggtgtgaaccca 301 ataaag

TSC18

TSC18 is a novel 66 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 15) 1 gaattcgaat cacgctcacc agccgcaacg tgaagtcgct 61ggagaaggtt tgtgcggact tgatca

TSC19

TSC19 is a novel 1613 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 16) 1CCAGCTCAGAGGTTCTAGGGGCAGCCGGCGCGCTTCTCTAGTTGCAGCTTGGGCGGCTCCTGTGGTGGGCGGCTAGGGGC81GAGCCGGGATGGGCTATAGACGCGCGACGTGATCAGTTCGCACGCGGACCCACGCCTCCCATCGCTCTGCCTCAAGAGCC161TATTCTGTGGGTGCAGGCACGCACCGGACGCAGACCCGGCCGGAGCATGCGGGGTGCGGTGTGGGCGGCCCGGAGGCGCG241CGGGGCAGCAGTGGCCTCGGTCCCCGGGCCCTGGGCCGGGTCCGCCCCCGCCGCCACCGCTGCTGTTGCTGCTACTACTG321CTGCTGGGCGGCGCGAGCGCTCAGTACTCCAGCGACCTGTGCAGCTGGAAGGGGAGTGGGCTCACCCGAGAGGCACGCAG401CAAGGAGGTGGAGCAGGTGTACCTGCGCTGCTCCGCAGGCTCTGTGGAGTGGATGTACCCAACTGGGGCGCTCATTGTTA481ACTACGGGCCCAACACCTTCTCACCTGCCCAGAACTTGACTGTGTGCATCAAGCCTTTCAGGCACTCCTCTGGAGCCAAT561ATTTATTTGGAAAAAACTGGAGAACTAAGACTGTTGGTGCGGGACATCAGAGGTGAGCCTGGCCAAGTGCAGTGCTTCAG641CCTGGAGCAGGGAGGCTTATTTGTGGAGGCGACACCCCAACAGGACATCAGCAGAAGGACCACAGGCTTCCAGTATGAGC721TGATGAGTGGGCAGAGGGGACTGGACCTGCACGTGCTGTCTGCCCCCTGTCGGCCTTGCAGTGACACTGAGGTCCTCCTT801GCCATCTGTACCAGTGACTTTGTTGTCCGAGGCTTCATTGAGGACGTCACACATGTACCAGAACAGCAAGTGTCAGTCAT881CTACCTGCGGGTGAACAGGCTTCACAGGCAGAAGAGCAGGGTCTTCCAGCCAGCTCCTGAGGACAGTGGCCACTGGCTGG961GCCATGTCACAACACTGCTGCAGTGTGGAGTACGACCAGGGCATGGGGAATTCCTCTTCACTGGACATGTGCACTTTGGG1041GAGGCACAACTTGGATGTGCCCCACGCTTTAGTGACTTTCAAAGGATGTACAGGAAAGCAGAAGAAATGGGCATAAACCC1121CTGTGAAATCAATATGGAGTGACTTGCAGGGTGACACAGTACTGTTGTCCTTCAGATGAGCCATGTTTTGTGGGCTCAGT1201CGCTCTATCATATCCTGATAGAGATTGCAGACTGGTGGCATGGGCCCAGCCTGGTGCTAGAACTGGGAAGGTACATGCTG1281TTCTGACCCCTTAGGTCCCAGCCAAGGATGCCCTGACCCATTGGAACTGCTGTAAAATGCAAACTAAGTTATTATATTTT1361TTTTGTAAAAGAAAAAAAAAAAAAAAAAAGAAAACTCCGCGCACAGGGGGGGTACGTCCCAATTCGCCAAAAACAGATGC1441TAGAACCCCTGGCGGCCCCCCCACCCCCACGGGAGACACTAGCTAACCAATTAATGCTTGGAAAATCCCTTCTGCACCGG1521TAGTACGAAAGGCCCACGATGCCTTCAAAGCTGCCTGGACGGAATGCAAATGAACGCTAATTTCTAATCCGGTAATTGTA1601 ACCGCATTCTACA

TSC20

TSC20 is a novel 2245 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 17) 1ACGTGACCGTGAGACCCTAGGAGCAATGGCGGGGCGGCTGGCTGGCTTCCTGATGTTGCTGGGGCTCGCGTCGCAGGGGC81CCGCGCCGGCATGTGCCGGGAAGATGAAGGTGGTGGAGGAGCCTAACACATTCGGGCTGAATAACCCGTTCTTGCCCCAG161GCAAGCCGCCTTCAGCCCAAGAGAGAGCCTTCAGCTGTATCCGGGCCCCTGCATCTCTTCAGACTTGCTGGCAAGTGCTT241TAGCCTAGTGGAGTCCACGTACAAGTATGAATTCTGCCCTTTCCACAACGTCACCCAGCACGAGCAGACCTTCCGCTGGA321ATGCCTACAGCGGGATCCTTGGCATCTGGCATGAGTGGGAAATCATCAACAATACCTTCAAGGGCATGTGGATGACTGAT401GGGGACTCCTGCCACTCCCGGAGCCGGCAGAGCAAGGTGGAGCTCACCTGTGGAAAGATCAACCGACTGGCCCACGTGTC481TGAGCCAAGCACCTGTGTCTATGCATTGACATTCGAGACCCCTCTTGTTTGCCATCCCCACTCTTTGTTAGTGTATCCAA561CTCTGTCAGAGGCCCTGCAGCAGCGCTGGGACCAGGTGGAACAGGACCTGGCAGATGAACTGATCACACCACAGGGCTAT641GAGAAGTTGCTAAGGGTACTTTTTCGAGGATGCCGGCTACTTAAAGGTCCCAGGAGAAACCCATCCCACCCAGCTGGCAG721GAGGTTCCAAGGGCCTAGGGCTTGAGACTCTGGACAACTGTAGAAAGGCACATGCAGAGCTGTCACAGGAGGTACAAAGA801CTGACGAGTCTGCTGCAACAGCATGGAATCCCCCACACTCAGCCCACAGAAACCACTCACTCTCAGCACCTGGGTCAGCA881GCTCCCCATAGGTGCAATCGCAGCAGAGCATCTGCGGAGTGACCCAGGACTACGTGGGAACATCCTGTGAGCAAGGTGGC961CACGAAGAATAGAAATATCCTGAGCTTTGAGTGTCCTTTCACAGAGTGAACAAAACTGGTGTGGTGTAGACACGGCTTCT1041TTTGGCATATTCTAGATCAGACAGTGTCACTGACAAACAAGAGGGACCTGCTGGCCAGCCTTTGTTGTGCCCAAAGATCC1121AGACAAAATAAAGATTCAAAGTTTTAATTAATTCCATACTGATAAAAAATAACTCCATGACTTCTGTAAACCATTGCATA1201AATGCTATTGTAAAAAAAATTAAACAAATGTTAACAACTTTAACAATTCACTAAAGTAAATGGTTATGTATTATAAATAT1281GACCATCTGGGTTAAGAAGATTCCATTCACATAACATTCTCAACTAATTTCTGAAGAACAAATGAACACAAAGGCTTCCA1361TAAGTTAATCCACATGCGCATCCATACTGGGGGAAGGCCTGCCAACCAGGTACACAAGACTCTGACACTACCATATACTG1441TTACTATTCAACACTAGAGAGTTAGACGACAACAGGCATCAGGACAGTGGTGGGTCCCAGTTCCTAGACCCATGGCCCCA1521CCTCCATTACCCACACACGGGCCTTAAGGCTCTCTCTCCCCTTCTTGGCCCTTCCCACCCAGGGTAGATCCTAGAAGCCT1601CAGCTCCTAAGAGGTCTGGAATGGATGGGAAAAGTGGCCCCTTCTGGGACGTTCTTTGGTCCTCCCCTGCACACCTGTCC1681TCAGAGCTCAGCCTGATTCCAGAAGAGCAGATGCTCAGGAAAGCTCCCCGCATGGGATGGGACCCAGGGTGCACTACCGC1761CTGCCTCCCCAGCCATCACAACAGCCCCAGAACTGCCCAGCCCCAGCCTGGAATGTCAGCCCAGGAGGAGTTAACCAGAG1841TAGCTTACATACAATCTAAAGCTTAATGTAACTGTATACAACTTGAAATTGTCCCGATGAGCTATCAATCACAAACACTG1921TCCTGTTACCACAGAGACCAAAAGCCTGACATGGGAAACAGTTCATAAATATGAATAAAAATAAACAATCTTAAACCATG2001GTAACAGTAGCACCAAATACACATGATCTAGGTACTGAGCTAATAAATCATTATCACTATAATTAAAAACAAAAGTCACT2081GAAATCAGGTCAATAGTTACCTTATTAAGTAGTGGGCTAGCTGTGGAATGTTGAAGATCCATTTCCTTTAAAATGATATA2161GGTCTTTTCTATCAGTTTGTCTTATATTAAAAAATGCTTTTAAATTTCCTACTATATTAAATACATTCTAATTTGGTCAC2241 TGATA

TSC21

TSC21 is a novel 171 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 18) 1 actagtcacc aaaatgcttg gttctaagtg gtagagaagg agacaccttagatataatac 61 aggtcaactt tttgacgtgg ggtgggggtg ggggtggggg tgggggtgaacatcacggtc 121 gcaaataagc agggtttgag ctttgtccag attgtagact taataaaatt y

TSC22

TSC22 is a novel 491 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 19) 1CAGTTGCAGAAGGGAGAAATCACGGCAGAATCATCGAGAAACCTGAAAAATGAGACCTAGAATGAAGTATTCCAACTCCA81AGATTTCCCCGGCAAAGTTCAGCAGCACCGCAGGCGAAGCCCTGGTCCCGCCTTGCAAAATAAGAAGATCCCAACATAAG161ACCAAAGAATTCTGCCATGTCTACTGCATGAGACTCCGTTCTGGCCTCACCATAAGAAAGGAGACTAGTTATTTTAGGAA241AGAACCCACGAAAAGATATTCACTAAAATCGGGTACCAAGCATGAAGAGAACTTCTCTGCCTATCCACGGGATTCTAGGA321AGAGATCCTTGCTTGGCAGTATCCAAGCATTTGCTGCGTCTGTTGACACATTGAGCATCCAAGGAACTTCACTTTTAACA401CAGTCTCCTGCCTCCCTGAGTACATACAATGACCAATCTGTTAGTTTTGTTTTGGAGAATGGATGTTATGTGATCAATGT481 TGACGACTCTG

TSC23

TSC23 is a novel 659 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 20) 1ATTTGGAATTTTAAGTTTTATCAATGCTTCTGGAAGCTTAGAACTGTACACGTGTGATGTCAGTCACATAGAGGAATGTG81CCCGGACTGCCTCATGCCTTTATTTTCCTTGGTAAATTTGAAGATAGAATGTCTGACTAGCGCAGTGACCAGAAAACAAT161GTGGTAGTCAACATCTCAGGCCATATTTTAAGATCCTGTAGAGCACTATTCATTTCAGGTTGCAGATGGAGTATTTTTGA241AACATCATTACTATGTAGATGCTTGGATAGGAGTGAGGGGGAGCTAGCAGATTTCCTGTGCCATTTATTCAGCTGATTGA321TGTACAGATGTAGGTTTATTTTGTAAAATCCACTGAAAGAATATGGCCACACCCTTGCCTACTTGATAGCATCAATACAG401AAGCCAAGAAGGACCACTAAGTAACCCCCTCTTCCCAGGGAGAGCAGCTAGCTTGAAATCTCTCGGATACAATCGATGCG481TCTGACCTTTGGGATCCTCACCATATGGGCAAACAATGGGCTTTGCAGGATGAGAGACACCCACTTAAACCTCTGACGAT561CTCGAATGGTTCATCTCTTCCGTCATTAACCAGTCATGGAAAACAATCAACAAACTCTGCCACGTGAAATATTTTTTCAG641 ACTTTTCTAACCCAAGCTT

TSC24

TSC24 is a novel 341 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 21) 1 raattcaaac aaagctttgg acaaggcccg gttaaaaagc aaagatgtcaagttggcaga 61 gactcatcag caggaatgct gccagaagtt tgaacagctt tctgaatctgcaaaagaaga 121 gctgataaac ttcaaacgga agagagtggc agcatttcga aagaacctaatcgaaatgtc 181 tgaactggaa ataaagcatg ccagaaacaa cgtctccctg ttgcagagctgcatcgactt 241 attcaagaac aactgacctg tctactctga aggacaccaa tgtgaaagccagcatcactt 301 gcacttaaat cattactgca aaagaaatag ctttgactag t

TSC25

TSC25 is a novel 53 bp gene fragment. The nucleic acid was initiallyidentified in a cloned fragment having the following sequence:

(SEQ ID NO: 22) 1 ggatcctgca aggctttggc cagctcagaa gcggcaaccc ctacacacctagg

General Methods

The TSCX nucleic acids and encoded polypeptides can be identified usingthe information provide above. In some embodiments, the TSCX nucleicacids and polypeptide correspond to nucleic acids or polypeptides whichinclude the various sequences (referenced by SEQ ID NOs) disclosed foreach TSCX polypeptide.

In its various aspects and embodiments, the invention includes providinga test cell population which includes at least one cell that is capableof expressing one or more of the sequences TSC 1-142. By “capable ofexpressing” is meant that the gene is present in an intact form in thecell and can be expressed. Expression of one, some, or all of the TSCXsequences is then detected, if present, and, preferably, measured. Usingsequence information provided by the database entries for the knownsequences, or the sequence information for the newly describedsequences, expression of the TSCX sequences can be detected (if present)and measured using techniques well known to one of ordinary skill in theart. For example, sequences within the sequence database entriescorresponding to TSCX sequences, or within the sequences disclosedherein, can be used to construct probes for detecting TSCX RNA sequencesin, e.g., northern blot hybridization analyses or methods whichspecifically, and, preferably, quantitatively amplify specific nucleicacid sequences. As another example, the sequences can be used toconstruct primers for specifically amplifying the TSCX sequences in,e.g., amplification-based detection methods such asreverse-transcription based polymerase chain reaction. When alterationsin gene expression are associated with gene amplification or deletion,sequence comparisons in test and reference populations can be made bycomparing relative amounts of the examined DNA sequences in the test andreference cell populations.

Expression can be also measured at the protein level, i.e., by measuringthe levels of polypeptides encoded by the gene products describedherein. Such methods are well known in the art and include, e.g.,immunoassays based on antibodies to proteins encoded by the genes.

Expression level of the TSCX sequences in the test cell population isthen compared to expression levels of the sequences in one or more cellsfrom a reference profile. Expression of sequences in test and controlpopulations of cells can be compared using any art-recognized method forcomparing expression of nucleic acid sequences. For example, expressioncan be compared using GENECALLING® methods as described in U.S. Pat. No.5,871,697 and in Shimkets et al., Nat. Biotechnol. 17:798-803. Invarious embodiments, the expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 35, 40, 50, 100, 150 or all of the sequences represented by TSC1-142 are measured. If desired, expression of these sequences can bemeasured along with other sequences whose expression is known to bealtered according to one of the herein described parameters orconditions.

A reference profile is an expression pattern derived from a singlereference population or from a plurality of expression patterns. Thereference profile can be a database of expression patterns frompreviously tested cells for which one of the herein-described conditions(e.g., tuberous sclerosis complex associated disorder) is known.Tuberous sclerosis complex assosiated disorders include for example,hamartomas, or hamartias in multiple organ systens, such as the brain,skin, heart or kidney, renal carcinoma, malignamnt angiomyolipoma,hypomelanotie macules, facila angiofibroma, shagreen patches and ungulafibromas.

In some embodiments, the test cell will be included in a cell samplefrom a subject known to contain, or to be suspected of having a tuberoussclerosis complex associated disorder. In other embodiments, the cellsample will be derived from a subject from a region known to contain, orsuspected of containing, a primary tumor, such as a renal carcinoma. Infurther embodiments, the cell sample will be derived from a subject froma region known to contain, or suspected of containing, a metastasis of aprimary tumor.

Preferably, cells in the reference profile are derived from a tissuetype as similar as possible to test cell, e.g., brain, skin, heart orkidney tissue. In some embodiments, the control cell is derived from thesame subject as the test cell, e.g., from a region proximal to theregion of origin of the test cell.

In some embodiments, the test cell population is compared to multiplereference profiles. Each of the multiple reference profiles may differin the known parameter or condition. Thus, a test cell population may becompared to a first reference profile known to have an tuberoussclerosis associated disorder, as well as a second reference populationknown not to have a tuberous sclerosis associated disorder.

In various embodiments, the expression of one or more sequences encodinggenes of expressed in distinct gene profiles, as listed in Table 1, iscompared. These gene profile include, e.g., “MEF and NSC−/− conserveddifferential expression” (such as, TSC 1-9), “MEF and NSC−/− oppositedifferential expression” (TSC 10-18), “NSC Only”, (TSC 19-44), and “MEFOnly” (TSC 45-57). In some embodiments, expression of members of two ormore gene profiles are compared.

Whether or not comparison of the gene expression profile in the testcell population to the reference profile reveals the presence, ordegree, of the measured condition depends on the composition of thereference profile. For example, if the profile is composed of cells thathave an tuberous sclerosis associated disorder, a similar geneexpression level in the test cell population and a reference profileindicates the presence of the tuberous sclerosis associated disorder inthe test cell population. Conversely, if the reference profile iscomposed of cells that do not have an tuberous sclerosis associateddisorder, a similar gene expression profile between the test cellpopulation and the reference profile indicates the absence of thetuberous sclerosis associated disorder in the test cell population

In various embodiments, the TSCX sequence in a test cell population isconsidered comparable in expression level to the expression level of theantileukoprotease sequence if its expression level varies within afactor of 2.0, 1.5, or 1.0 fold to the level of the TSCX transcript inthe reference profile. In various embodiments, a TSC sequence in a testcell population can be considered altered in levels of expression if itsexpression level varies from the reference cell population by more than1.0, 1.5, 2.0 or more fold from the expression level of thecorresponding antileukoprotease sequence in the reference cellpopulation.

If desired, comparison of differentially expressed sequences between atest cell population and a reference profile can be done with respect toa control nucleic acid whose expression is independent of the parameteror condition being measured. Expression levels of the control nucleicacid in the test and reference nucleic acid can be used to normalizesignal levels in the compared populations.

The test cell population can be any number of cells, i.e., one or morecells, and can be provided in vitro, in vivo, or ex vivo.

In other embodiments, the test cell population can be divided into twoor more subpopulations. The subpopulations can be created by dividingthe first population of cells to create as identical a subpopulation aspossible. This will be suitable, in, for example, in vitro or ex vivoscreening methods. In some embodiments, various sub populations can beexposed to a control agent, and/or a test agent, multiple test agents,or, e.g., varying dosages of one or multiple test agents administeredtogether, or in various combinations.

The subject is preferably a mammal. The mammal can be, e.g., a human,non-human primate, mouse, rat, dog, cat, horse, or cow.

Diagnosing a Tuberous Sclerosis Complex Associated Disorder

The invention provides a method of diagnosing or determining thesusceptibility of a tuberous sclerosis complex associated disorder,e.g., hamartomas, or hamartias in multiple organ systens, such as thebrain, skin, heart or kidney, renal carcinoma, malignant angiomyolipoma,hypomelanotic macules, facila angiofibroma, shagreen patches and ungulafibromas. A tuberous sclerosis complex associated disorder is diagnosedby examining the expression of a nucleic acid encoding a TSCX nucleicacid from a test population of cells from a subject suspected of havinga tuberous sclerosis complex associated disorder. The population ofcells may contain cells of the brain, or may alternatively may containcells the eye, skin, heart, or kidney.

Expression of a TSCX nucleic acid is measured in the test cell andcompared to the expression of the sequence in the reference profile. Areference profile can be a TSC disorder positive reference profile. By“TSC disorder positive reference profile” is meant that the referenceprofile contains cells derived from tissues with a tuberous sclerosiscomplex associated disorder. Alternatively, the reference profile can bean TSC disorder negative reference profile. By “TSC negative referenceprofile” is meant that the reference profile contains cells derived fromtissues without a tuberous sclerosis complex associated disorder.

When a reference profile is an TSC disorder positive reference profile,a similarity in expression between TSCX sequences in the test populationand the reference profile indicates the presence of a tuberous sclerosiscomplex associated disorder in the subject. Conversely, a difference inexpression in the test cell population between TSCX sequences in thetest population and the TSC disorder positive reference profileindicates the absence of a tuberous sclerosis complex associateddisorder in the subject.

When the reference profile is TSC disorder negative reference profile,an difference in expression pattern between the test cell population andthe TSC disorder negative reference profile indicates the presence of atuberous sclerosis complex associated disorder. Conversely, a similarityin expression between TSCX sequences in the test population and the TSCdisorder negative reference profile indicates the absence of a tuberoussclerosis complex associated disorder in the subject.

Methods of Treating Disorders Associated with Tuberous Sclerosis Complex

The invention provides a method for treating tuberous sclerosis complexassociated disorders in a subject by administering to a subject in needthereof a compound that modulates the expression of one or more TSCXnucleic acids or polypeptides. Administration can be prophylactic ortherapeutic to a subject at risk of (or susceptible to) tuberoussclerosis complex associated disorder. The tuberous sclerosis associateddisorder can be, e.g., hamartomas, or hamartias in multiple organsystens, such as the brain, skin, heart or kidney, renal carcinoma,malignant angiomyolipoma, hypomelanotic macules, facila angiofibroma,shagreen patches and ungula fibromas.

The therapeutic method includes decreasing or inhibiting the expression,or function, or TSCX nucleic acids in the diseased cell relative tonormal cells of the tissue type from which the diseased cells arederived. In these methods, the subject is treated with an effectiveamount of a compound, which decreases the amount of a TSCX nucleic acidor polypeptide in the subject. Administration can be systemic or local,e.g., in the immediate vicinity of, the subject's diseased cells.Expression can be inhibited in any of several ways known in the art. Forexample, expression can be inhibited by administering to the subject anucleic acid that inhibits, or antagonizes, the expression of the TSCX.In one embodiment, an antisense oligonucleotide can be administeredwhich disrupts expression of a TSCX nucleic acid.

Alternatively, the function a TSCX can be inhibited by administering acompound that binds to or otherwise inhibits the function of the TSCXgene products. The compound can be, e.g., an antibody to a polypeptideencoded by a TSCX nucleic acid.

These modulatory methods can be performed ex vivo or in vitro (e.g., byculturing the cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant expression or activity TSCX proteinsor nucleic acid molecules. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or combination of agents that modulates (e.g.,upregulates or downregulates) expression or activity of TSCX nucleicacids or polypeptides. In another embodiment, the method involvesadministering a protein or combination of proteins or a nucleic acidmolecule or combination of nucleic acid, molecules as therapy tocompensate for aberrant expression or activity of a TSCX nucleic acid.

Therapeutics that may be utilized include, e.g., (i) a polypeptide, oranalogs, derivatives, fragments or homologs thereof of the overexpressedsequence; (ii) antibodies to the overexpressed sequence; (iii) antisensenucleic acids or nucleic acids that are “dysfunctional” (i.e., due to aheterologous insertion within the coding sequences of coding sequencesof one or more overexpressed or underexpressed sequences); or (v)modulators (i.e., inhibitors, agonists and antagonists that alter theinteraction between an overexpressed polypeptide and its bindingpartner. The dysfunctional antisense molecules are utilized to“knockout” endogenous function of a polypeptide by homologousrecombination (see, e.g., Capecchi, Science 244: 1288-1292 1989)

Increased or decreased levels can be readily detected by quantifyingpeptide and/or RNA, by obtaining a patient tissue sample (e.g., frombiopsy tissue) and assaying it in vitro for RNA or peptide levels,structure and/or activity of the expressed peptides (or mRNAs of a genewhose expression is altered). Methods that are well-known within the artinclude, but are not limited to, immunoassays (e.g., by Western blotanalysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/orhybridization assays to detect expression of mRNAs (e.g., Northernassays, dot blots, in situ hybridization, etc.).

Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of aberrant gene expression,such that a disease or disorder is prevented or, alternatively, delayedin its progression. Depending on the type of aberrant expressiondetected, the agent can be used for treating the subject. Theappropriate agent can be determined based on screening assays describedherein.

Screening Assays for Identifying a Candidate Therapeutic Agent forTreating or Preventing Tuberous Sclerosis Associated Disorder

The differentially expressed sequences disclosed herein can also be usedto identify candidate therapeutic agents to treat or prevent tuberoussclerosis associated disorders. The therapeutic agent can be identifiedby providing a cell population that includes cells capable of expressingTSCX nucleic acids. Expression of the nucleic acid sequences in the testcell population is then compared to the expression of the nucleic acidsequences in a reference cell population, which is a cell populationthat has not been exposed to the test agent, or, in some embodiments, acell population exposed the test agent. Comparison can be performed ontest and reference samples measured concurrently or at temporallydistinct times. An example of the latter is the use of compiledexpression information, e.g., a sequence database, which assemblesinformation about expression levels of known sequences followingadministration of various agents. For example, alteration of expressionlevels following administration of test agent can be compared to theexpression changes observed in the nucleic acid sequences followingadministration of a control agent.

An decrease in expression of the nucleic acid sequence in the test cellpopulation compared to the expression of the nucleic acid sequence inthe reference cell population that has not been exposed to the testagent indicates the test agent is a canidate therapeutic agent.

The test agent can be a compound not previously described or can be apreviously known compound but which is not known to be an agent fortreating tuberous sclerosis complex disorders.

The invention also includes a compound identified according to thisscreening method.

An agent effective in stimulating expression of underexpressed genes, orin suppressing expression of overexpressed genes can be further testedfor its ability to prevent the tuberous sclerosis complex associateddisorders, and as a potential therapeutic useful for the treatment ofsuch pathophysiology. Further evaluation of the clinical usefulness ofsuch a compound can be performed using standard methods of evaluatingtoxicity and clinical effectiveness.

Selecting a Therapeutic Agent for Treating Tuberous Sclerosis ComplexAssociated Disorder that is Appropriate for a Particular Individual

Differences in the genetic makeup of individuals can result indifferences in their relative abilities to metabolize various drugs. Anagent that is metabolized in a subject to act as a therapeutic agent canmanifest itself by inducing a change in gene expression pattern fromthat characteristic of a pathophysiologic state to a gene expressionpattern characteristic of a non-pathophysiologic state. Accordingly, thedifferentially expressed TSCX sequences disclosed herein allow for aputative therapeutic or prophylactic agent to be tested in a test cellpopulation from a selected subject in order to determine if the agent isa suitable therapeutic agent in the subject.

To identify a therapeutic agent, that is appropriate for a specificsubject, a test cell population from the subject is exposed to atherapeutic agent, and the expression of one or more of TSCX 1-141.

In some embodiments, the agent is first mixed with a cell extract, whichcontains enzymes that metabolize drugs into an active form. Theactivated form of the therapeutic agent can then be mixed with the testcell population and gene expression measured. Preferably, the cellpopulation is contacted ex vivo with the agent or activated form of theagent.

Expression of the nucleic acid sequences in the test cell population isthen compared to the expression of the nucleic acid sequences areference cell population. The reference cell population includes atleast one cell whose tuberous sclerosis complex status is known. By“tuberous sclerosis complex status is meant, whether or not thereference cell population contains cells known to have tuberoussclerosis complex subject.

The test agent can be any compound or composition.

Assessing Efficacy of Treatment of a Tuberous Sclerosis ComplexAssociated Disorder in a Subject

The differentially expressed TSCX sequences identified herein also allowfor the course of treatment of a tuberous sclerosis complex associateddisorder to be monitored. In this method, a test cell population isprovided from a subject undergoing treatment for a tuberous sclerosiscomplex associated disorder. If desired, test cell populations can betaken from the subject at various time points before, during, or aftertreatment. Expression of one or more of the TSCX sequences, e.g., TSCXs:1-142, in the cell population is then measured and compared to areference cell population which includes cells whose pathophysiologicstate is known. Preferably, the reference cells not been exposed to thetreatment.

If the reference cell population contains no cells exposed to thetreatment, a similarity in expression between TSCX sequences in the testcell population and the reference cell population indicates that thetreatment is efficacious. However, a difference in expression betweenTSCX sequences in the test population and this reference cell populationindicates the treatment is not efficacious.

By “efficacious” is meant that the treatment leads to a decrease in thepathophysiology in a subject. When treatment is appliedprophylactically, “efficacious” means that the treatment retards orprevents a pathophysiology.

Efficaciousness can be determined in association with any known methodfor treating the particular pathophysiology

Assessing the Prognosis of a Subject with a Tuberous Sclerosis ComplexAssociated Disorder

Also provided is a method of assessing the prognosis of a subject with atuberous sclerosis complex associated disorder by comparing theexpression of a TSCX nucleic acid in a test cell population to theexpression of the sequences in a reference profile derived from patientsover a spectrum of disease stages. By comparing gene expression of aTSCX nucleic acid in the test cell population and the reference profile,or by comparing the pattern of gene expression overtime in test cellpopulations derived from the subject, the prognosis of the subject canbe assessed.

The reference profile includes primarily noncancerous or cancerouscells. A reference profile which includes primarily noncancerous cellsis a non-cancer reference profile. A reference profile which includesprimarily cancerous cells is a cancer reference profile. In someembodiments the cancer reference profile includes primarily disseminatedcancerous cells. When the reference profile includes primarilynoncancerous cells, an increase of expression of TSCX nucleic acids inthe test cell population, indicates less favorable prognosis.Conversely, when the reference profile includes primarily cancerouscells, an decrease of expression of TSCX nucleic acids in the test cellpopulation, indicates more favorable prognosis.

Pharmaceutical Compositions

In another aspect the invention includes pharmaceutical, or therapeutic,compositions containing one or more therapeutic compounds describedherein. Pharmaceutical formulations may include those suitable for oral,rectal, nasal, topical (including buccal and sub-lingual), vaginal orparenteral (including intramuscular, sub-cutaneous and intravenous)administration, or for administration by inhalation or insufflation. Theformulations may, where appropriate, be conveniently presented indiscrete dosage units and may be prepared by any of the methods wellknown in the art of pharmacy. All such pharmacy methods include thesteps of bringing into association the active compound with liquidcarriers or finely divided solid carriers or both as needed and then, ifnecessary, shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration mayconveniently be presented as discrete units, such as capsules, cachetsor tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; or as a solution, a suspension oras an emulsion. The active ingredient may also be presented as a boluselectuary or paste, and be in a pure form, i.e., without a carrier.Tablets and capsules for oral administration may contain conventionalexcipients such as binding agents, fillers, lubricants, disintegrant orwetting agents. A tablet may be made by compression or molding,optionally with one or more formulational ingredients. Compressedtablets may be prepared by compressing in a suitable machine the activeingredients in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, lubricating,surface active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may be coatedaccording to methods well known in the art. Oral fluid preparations maybe in the form of for example, aqueous or oily suspensions, solutions,emulsions, syrups or elixirs, or may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may contain conventional additives such assuspending agents, emulsifying agents, non-aqueous vehicles (which mayinclude edible oils), or preservatives. The tablets may optionally beformulated so as to provide slow or controlled release of the activeingredient therein.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, saline, water-for-injection,immediately prior to use. Alternatively, the formulations may bepresented for continuous infusion. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtablets of the kind previously described.

Formulations for rectal administration may be presented as a suppositorywith the usual carriers such as cocoa butter or polyethylene glycol.Formulations for topical administration in the mouth, for examplebuccally or sublingually, include lozenges, comprising the activeingredient in a flavored base such as sucrose and acacia or tragacanth,and pastilles comprising the active ingredient in a base such as gelatinand glycerin or sucrose and acacia. For intra-nasal administration thecompounds of the invention may be used as a liquid spray or dispersiblepowder or in the form of drops. Drops may be formulated with an aqueousor non-aqueous base also comprising one or more dispersing agents,solubilizing agents or suspending agents. Liquid sprays are convenientlydelivered from pressurized packs.

For administration by inhalation the compounds are convenientlydelivered from an insufflator, nebulizer, pressurized packs or otherconvenient means of delivering an aerosol spray. Pressurized packs maycomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichiorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecompounds may take the form of a dry powder composition, for example apowder mix of the compound and a suitable powder base such as lactose orstarch. The powder composition may be presented in unit dosage form, infor example, capsules, cartridges, gelatin or blister packs from whichthe powder may be administered with the aid of an inhalator orinsuffiator.

When desired, the above described formulations, adapted to givesustained release of the active ingredient, may be employed. Thepharmaceutical compositions may also contain other active ingredientssuch as antimicrobial agents, immunosuppressants or preservatives.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude flavoring agents.

Preferred unit dosage formulations are those containing an effectivedose, as recited below, or an appropriate fraction thereof, of theactive ingredient.

For each of the aforementioned conditions, the compositions may beadministered orally or via injection at a dose of from about 0.1 toabout 250 mg/kg per day. The dose range for adult humans is generallyfrom about 5 mg to about 17.5 g/day, preferably about 5 mg to about 10g/day, and most preferably about 100 mg to about 3 g/day. Tablets orother unit dosage forms of presentation provided in discrete units mayconveniently contain an amount which is effective at such dosage or as amultiple of the same, for instance, units containing about 5 mg to about500 mg, usually from about 100 mg to about 500 mg.

The pharmaceutical composition preferably is administered orally or byinjection (intravenous or subcutaneous), and the precise amountadministered to a subject will be the responsibility of the attendantphysician. However, the dose employed will depend upon a number offactors, including the age and sex of the subject, the precise disorderbeing treated, and its severity. Also the route of administration mayvary depending upon the condition and its severity.

TSCX Nucleic Acids

Also provided in the invention are novel nucleic acid comprising anucleic acid sequence selected from the group consisting of TSC: 1-8,10-12, and 15-25 (SEQ ID NO: 1-22) or its complement, as well as vectorsand cells including these nucleic acids.

Thus, one aspect of the invention pertains to isolated TSCX nucleic acidmolecules that encode TSCX proteins or biologically active portionsthereof. Also included are nucleic acid fragments sufficient for use ashybridization probes to identify TSCX-encoding nucleic acids (e.g., TSCXmRNA) and fragments for use as polymerase chain reaction (PCR) primersfor the amplification or mutation of TSCX nucleic acid molecules. Asused herein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA),analogs of the DNA or RNA generated using nucleotide analogs, andderivatives, fragments and homologs thereof. The nucleic acid moleculecan be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

“Probes” refer to nucleic acid sequences of variable length, preferablybetween at least about 10 nucleotides (nt) or as many as about, e.g.,6,000 nt, depending on use. Probes are used in the detection ofidentical, similar, or complementary nucleic acid sequences. Longerlength probes are usually obtained from a natural or recombinant source,are highly specific and much slower to hybridize than oligomers. Probesmay be single- or double-stranded and designed to have specificity in.PCR, membrane-based hybridization technologies, or ELISA-liketechnologies.

An “isolated” nucleic acid molecule is one that is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. Examples of isolated nucleic acid molecules include, butare not limited to, recombinant DNA molecules contained in a vector,recombinant DNA molecules maintained in a heterologous host cell,partially or substantially purified nucleic acid molecules, andsynthetic DNA or RNA molecules. Preferably, an “isolated” nucleic acidis free of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated TSCX nucleic acid moleculecan contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or 0.1 kb of nucleotide sequences which naturally flank thenucleic acid molecule in genomic DNA of the cell from which the nucleicacid is derived. Moreover, an “isolated” nucleic acid molecule, such asa cDNA molecule, can be substantially free of other cellular material orculture medium when produced by recombinant techniques, or of chemicalprecursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of any of TSC: 1-8, 10-12, and15-25, or a complement of any of these nucleotide sequences, can beisolated using standard molecular biology techniques and the sequenceinformation provided herein. Using all or a portion of these nucleicacid sequences as a hybridization probe, TSCX nucleic acid sequences canbe isolated using standard hybridization and cloning techniques (e.g.,as described in Sambrook et al., eds., MOLECULAR CLONING: A LABORATORYMANUAL 2^(nd) Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; and Ausubel, et al., eds., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993.)

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to TSCX nucleotide sequencescan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

As used herein, the term “oligonucleotide” refers to a series of linkednucleotide residues, which oligonucleotide has a sufficient number ofnucleotide bases to be used in a PCR reaction. A short oligonucleotidesequence may be based on, or designed from, a genomic or cDNA sequenceand is used to amplify, confirm, or reveal the presence of an identical,similar or complementary DNA or RNA in a particular cell or tissue.Oligonucleotides comprise portions of a nucleic acid sequence having atleast about 10 nt and as many as 50 nt, preferably about 15 nt to 30 nt.They may be chemically synthesized and may be used as probes.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule that is a complement of thenucleotide sequence shown in TSCX::1-7, 10-13, 19-34, 45-53, 58-85,111-113, 120, 130, 132-134 and 138. In another embodiment, an isolatednucleic acid molecule of the invention comprises a nucleic acid moleculethat is a complement of the nucleotide sequence shown in any of thesesequences, or a portion of any of these nucleotide sequences. A nucleicacid molecule that is complementary to the nucleotide sequence shown inTSC: 1-8, 10-12, and 15-25 is one that is sufficiently complementary tothe nucleotide sequence shown, such that it can hydrogen bond withlittle or no mismatches to the nucleotide sequences shown, therebyforming a stable duplex.

As used herein, the term “complementary” refers to Watson-Crick orHoogsteen base pairing between nucleotides units of a nucleic acidmolecule, and the term “binding” means the physical or chemicalinteraction between two polypeptides or compounds or associatedpolypeptides or compounds or combinations thereof. Binding includesionic, non-ionic, Von der Waals, hydrophobic interactions, etc. Aphysical interaction can be either direct or indirect. Indirectinteractions may be through or due to the effects of another polypeptideor compound. Direct binding refers to interactions that do not takeplace through, or due to, the effect of another polypeptide or compound,but instead are without other substantial chemical intermediates.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of TSC: 1-8, 10-12, and 15-25 e.g.,a fragment that can be used as a probe or primer or a fragment encodinga biologically active portion of TSCX. Fragments provided herein aredefined as sequences of at least 6 (contiguous) nucleic acids or atleast 4 (contiguous) amino acids, a length sufficient to allow forspecific hybridization in the case of nucleic acids or for specificrecognition of an epitope in the case of amino acids, respectively, andare at most some portion less than a full length sequence. Fragments maybe derived from any contiguous portion of a nucleic acid or amino acidsequence of choice. Derivatives are nucleic acid sequences or amino acidsequences formed from the native compounds either directly or bymodification or partial substitution. Analogs are nucleic acid sequencesor amino acid sequences that have a structure similar to, but notidentical to, the native compound but differs from it in respect tocertain components or side chains. Analogs may be synthetic or from adifferent evolutionary origin and may have a similar or oppositemetabolic activity compared to wild type.

Derivatives and analogs may be full length or other than full length, ifthe derivative or analog contains a modified nucleic acid or amino acid,as described below. Derivatives or analogs of the nucleic acids orproteins of the invention include, but are not limited to, moleculescomprising regions that are substantially homologous to the nucleicacids or proteins of the invention, in various embodiments, by at leastabout 45%, 50%, 70%, 80%, 95%, 98%, or even 99% identity (with apreferred identity of 80-99%) over a nucleic acid or amino acid sequenceof identical size or when compared to an aligned sequence in which thealignment is done by a computer homology program known in the art, orwhose encoding nucleic acid is capable of hybridizing to the complementof a sequence encoding the aforementioned proteins under stringent,moderately stringent, or low stringent conditions. See e.g. Ausubel, etal., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NewYork, N.Y., 1993, and below. An exemplary program is the Gap program(Wisconsin Sequence Analysis Package, Version 8 for UNIX, GeneticsComputer Group, University Research Park, Madison, Wis.) using thedefault settings, which uses the algorithm of Smith and Waterman (Adv.Appl. Math., 1981, 2: 482-489, which in incorporated herein by referencein its entirety).

A “homologous nucleic acid sequence” or “homologous amino acidsequence,” or variations thereof, refer to sequences characterized by ahomology at the nucleotide level or amino acid level as discussed above.Homologous nucleotide sequences encode those sequences coding forisoforms of a TSCX polypeptide. Isoforms can be expressed in differenttissues of the same organism as a result of for example, alternativesplicing of RNA. Alternatively, isoforms can be encoded by differentgenes. In the present invention, homologous nucleotide sequences includenucleotide sequences encoding for a TSCX polypeptide of species otherthan humans, including, but not limited to, mammals, and thus caninclude, e.g., mouse, rat, rabbit, dog, cat cow, horse, and otherorganisms. Homologous nucleotide sequences also include, but are notlimited to, naturally occurring allelic variations and mutations of thenucleotide sequences set forth herein. A homologous nucleotide sequencedocs not, however, include the nucleotide sequence encoding a human TSCXprotein. Homologous nucleic acid sequences include those nucleic acidsequences that encode conservative amino acid substitutions (see below)in a TSCX polypeptide, as well as a polypeptide having a TSCX activity.A homologous amino acid sequence does not encode the amino acid sequenceof a human TSCX polypeptide.

The nucleotide sequence determined from the cloning of human TSCX genesallows for the generation of probes and primers designed for use inidentifying and/or cloning TSCX homologues in other cell types, e.g.,from other tissues, as well as TSCX homologues from other mammals. Theprobe/primer typically comprises a substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutivesense strand nucleotide sequence of a nucleic acid comprising a TSCXsequence, or an anti-sense strand nucleotide sequence of a nucleic acidcomprising a TSCX sequence, or of a naturally occurring mutant of thesesequences.

Probes based on human TSCX nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In various embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a TSCX protein, such as by measuring a level ofa TSCX-encoding nucleic acid in a sample of cells from a subject e.g.,detecting TSCX mRNA levels or determining whether a genomic TSCX genehas been mutated or deleted.

“A polypeptide having a biologically active portion of TSCX” refers topolypeptides exhibiting activity similar, but not necessarily identicalto, an activity of a polypeptide of the present invention, includingmature forms, as measured in a particular biological assay, with orwithout dose dependency. A nucleic acid fragment encoding a“biologically active portion of TSCX” can be prepared by isolating aportion of TSC: 1-8, 10-12, and 15-25, that encodes a polypeptide havinga TSCX biological activity, expressing the encoded portion of TSCXprotein (e.g., by recombinant expression in vitro) and assessing theactivity of the encoded portion of TSCX. For example, a nucleic acidfragment encoding a biologically active portion of a TSCX polypeptidecan optionally include an ATP-binding domain. In another embodiment, anucleic acid fragment encoding a biologically active portion of TSCXincludes one or more regions.

TSCX Variants

The invention further encompasses nucleic acid molecules that differfrom the disclosed or referenced TSCX nucleotide sequences due todegeneracy of the genetic code.

These nucleic acids thus encode the same TSCX protein as that encoded bynucleotide sequence comprising a TSCX nucleic acid as shown in, e.g.,TSC: 1-8, 10-12, and 15-25

In addition to the rat TSCX nucleotide sequence shown in TSC: 1-8,10-12, and 15-25, it will be appreciated by those skilled in the artthat DNA sequence polymorphisms that lead to changes in the amino acidsequences of a TSCX polypeptide may exist within a population (e.g., thehuman population). Such genetic polymorphism in the TSCX gene may existamong individuals within a population due to natural allelic variation.As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a TSCX protein,preferably a mammalian TSCX protein. Such natural allelic variations cantypically result in 1-5% variance in the nucleotide sequence of the TSCXgene. Any and all such nucleotide variations and resulting amino acidpolymorphisms in TSCX that are the result of natural allelic variationand that do not alter the functional activity of TSCX are intended to bewithin the scope of the invention.

Moreover, nucleic acid molecules encoding TSCX proteins from otherspecies, and thus that have a nucleotide sequence that differs from thehuman sequence of TSC: 1-8, 10-12, and 15-25, are intended to be withinthe scope of the invention. Nucleic acid molecules corresponding tonatural allelic variants and homologues of the TSCX DNAs of theinvention can be isolated based on their homology to the human TSCXnucleic acids disclosed herein using the human cDNAs, or a portionthereof, as a hybridization probe according to standard hybridizationtechniques under stringent hybridization conditions. For example, asoluble human TSCX DNA can be isolated based on its homology to humanmembrane-bound TSCX. Likewise, a membrane-bound human TSCX DNA can beisolated based on its homology to soluble human TSCX.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence of TSC: 1-8, 10-12, and 15-25. In anotherembodiment, the nucleic acid is at least 10, 25, 50, 100, 250 or 500nucleotides in length. In another embodiment, an isolated nucleic acidmolecule of the invention hybridizes to the coding region. As usedherein, the term “hybridizes under stringent conditions” is intended todescribe conditions for hybridization and washing under which nucleotidesequences at least 60% homologous to each other typically remainhybridized to each other.

Homologs (i.e., nucleic acids encoding TSCX proteins derived fromspecies other than human) or other related sequences (e.g., paralogs)can be obtained by low, moderate or high stringency hybridization withall or a portion of the particular human sequence as a probe usingmethods well known in the art for nucleic acid hybridization andcloning.

As used herein, the phrase “stringent hybridization conditions” refersto conditions under which a probe, primer or oligonucleotide willhybridize to its target sequence, but to no other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures than shorter sequences. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. Since thetarget sequences are generally present at excess, at Tm, 50% of theprobes are occupied at equilibrium. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes,primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about60° C. for longer probes, primers and oligonucleotides. Stringentconditions may also be achieved with the addition of destabilizingagents, such as formamide.

Stringent conditions are known to those skilled in the art and can befound in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequencesat least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous toeach other typically remain hybridized to each other. A non-limitingexample of stringent hybridization conditions is hybridization in a highsalt buffer comprising 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02%PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm. DNAat 65° C. This hybridization is followed by one or more washes in0.2×SSC, 0.01% BSA at 50° C. An isolated nucleic acid molecule of theinvention that hybridizes under stringent conditions to the sequence ofTSC: 1-8, 10-12, and 15-25 corresponds to a naturally occurring nucleicacid molecule. As used herein, a “naturally-occurring” nucleic acidmolecule refers to an RNA or DNA molecule having a nucleotide sequencethat occurs in nature (e.g., encodes a natural protein).

In a second embodiment, a nucleic acid sequence that is hybridizable tothe nucleic acid molecule comprising the nucleotide sequence of TSC:1-8, 10-12, and 15-25 or fragments, analogs or derivatives thereof,under conditions of moderate stringency is provided. A non-limitingexample of moderate stringency hybridization conditions arehybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 mg/mldenatured salmon sperm DNA at 55° C., followed by one or more washes in1×SSC, 0.1% SDS at 37° C. Other conditions of moderate stringency thatmay be used are well known in the art. See, e.g., Ausubel et al. (eds.),(eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons,NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORYMANUAL, Stockton Press,

NY.

In a third embodiment, a nucleic acid that is hybridizable to thenucleic acid molecule comprising the nucleotide sequence of TSC: 1-8,10-12, and 15-25 or fragments, analogs or derivatives thereof, underconditions of low stringency, is provided. A non-limiting example of lowstringency hybridization conditions are hybridization in 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2%BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfateat 40° C., followed by one or more washes in 2×SSC, 25 mM Tris-HCl (pH7.4), 5 mM EDTA, and 0.1% SDS at 50° C. Other conditions of lowstringency that may be used are well known in the art (e.g., as employedfor cross-species hybridizations). See, e.g., Ausubel et al. (eds.),1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, andKriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,Stockton Press, NY; Shilo et al., 1981, Proc Nati Acad Sci USA 78:6789-6792.

Conservative Mutations

In addition to naturally-occurring allelic variants of the TSCX sequencethat may exist in the population, the skilled artisan will furtherappreciate that changes can be introduced into an TSCX nucleic acid ordirectly into an TSCX polypeptide sequence without altering thefunctional ability of the TSCX protein. In some embodiments, thenucleotide sequence of TSC: 1-8, 10-12, and 15-25 will be altered,thereby leading to changes in the amino acid sequence of the encodedTSCX protein. For example, nucleotide substitutions that result in aminoacid substitutions at various “non-essential” amino acid residues can bemade in the sequence of TSC: 1-8, 10-12, and 15-25. A “non-essential”amino acid residue is a residue that can be altered from the wild-typesequence of TSCX without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity. Forexample, amino acid residues that are conserved among the TSCX proteinsof the present invention, are predicted to be particularly unamenable toalteration.

In addition, amino acid residues that are conserved among family membersof the TSCX proteins of the present invention, are also predicted to beparticularly unamenable to alteration. As such, these conserved domainsare not likely to be amenable to mutation. Other amino acid residues,however, (e.g., those that are not conserved or only semi-conservedamong members of the TSCX proteins) may not be essential for activityand thus are likely to be amenable to alteration.

Another aspect of the invention pertains to nucleic acid moleculesencoding TSCX proteins that contain changes in amino acid residues thatare not essential for activity. Such TSCX proteins differ in amino acidsequence from the amino acid sequences of polypeptides encoded bynucleic acids containing TSC: 1-8, 10-12, and 15-25, yet retainbiological activity. In one embodiment, the isolated nucleic acidmolecule comprises a nucleotide sequence encoding a protein, wherein theprotein comprises an amino acid sequence at least about 45% homologous,more preferably 60%, and still more preferably at least about 70%, 80%,90%, 95%, 98%, and most preferably at least about 99% homologous to theamino acid sequence of the amino acid sequences of polypeptides encodedby nucleic acids comprising TSC: 1-8, 10-12, and 15-25.

An isolated nucleic acid molecule encoding a TSCX protein homologous tocan be created by introducing one or more nucleotide substitutions,additions or deletions into the nucleotide sequence of a nucleic acidcomprising TSC: 1-8, 10-12, and 15-25, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein.

Mutations can be introduced into a nucleic acid comprising TSC: 1-8,10-12, and 15-25 by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. Preferably, conservative aminoacid substitutions are made at one or more predicted non-essential aminoacid residues. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in TSCX isreplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a TSCX coding sequence, such asby saturation mutagenesis, and the resultant mutants can be screened forTSCX biological activity to identify mutants that retain activity.Following mutagenesis of the nucleic acid, the encoded protein can beexpressed by any recombinant technology known in the art and theactivity of the protein can be determined.

In one embodiment, a mutant TSCX protein can be assayed for (1) theability to form protein:protein interactions with other TSCX proteins,other cell-surface proteins, or biologically active portions thereof,(2) complex formation between a mutant TSCX protein and a TSCX ligand;(3) the ability of a mutant TSCX protein to bind to an intracellulartarget protein or biologically active portion thereof; (e.g., avidinproteins); (4) the ability to bind ATP; or (5) the ability tospecifically bind a TSCX protein antibody.

In other specific embodiments, the nucleic acid is RNA or DNA. Thefragment or the fragment of the complementary polynucleotide sequence isbetween about 10 and about 100 nucleotides in length, e.g., betweenabout 10 and about 90 nucleotides in length, or about 10 and about 75nucleotides in length, about 10 and about 50 bases in length, about 10and about 40 bases in length, or about 15 and about 30 bases in length.

Antisense

Another aspect of the invention pertains to isolated antisense nucleicacid molecules that are hybridizable to or complementary to the nucleicacid molecule comprising the nucleotide sequence of a TSCX sequence orfragments, analogs or derivatives thereof. An “antisense” nucleic acidcomprises a nucleotide sequence that is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. In specific aspects, antisense nucleic acid molecules areprovided that comprise a sequence complementary to at least about 10,25, 50, 100, 250 or 500 nucleotides or an entire TSCX coding strand, orto only a portion thereof. Nucleic acid molecules encoding fragments,homologs, derivatives and analogs of a TSCX protein, or antisensenucleic acids complementary to a nucleic acid comprising a TSCX nucleicacid sequence are additionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence encodingTSCX. The term “coding region” refers to the region of the nucleotidesequence comprising codons which are translated into amino acidresidues. In another embodiment, the antisense nucleic acid molecule isantisense to a “noncoding region” of the coding strand of a nucleotidesequence encoding TSCX. The term “noncoding region” refers to 5′ and 3′sequences which flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding TSCX disclosed herein,antisense nucleic acids of the invention can be designed according tothe rules of Watson and Crick or Hoogsteen base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof TSCX mRNA, but more preferably is an oligonucleotide that isantisense to only a portion of the coding or noncoding region of TSCXmRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of TSCX mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid ofthe invention can be constructed using chemical synthesis or enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused.

Examples of modified nucleotides that can be used to generate theantisense nucleic acid include: 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a TSCX proteinto thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule that binds toDNA duplexes, through specific interactions in the major groove of thedouble helix. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies that bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of antisense molecules,vector constructs in which the antisense nucleic acid molecule is placedunder the control of a strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). Theantisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett215: 327-330).

Ribozymes and PNA Moieties

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity that are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveTSCX mRNA transcripts to thereby inhibit translation of TSCX mRNA. Aribozyme having specificity for a TSCX-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a TSCX DNA disclosedherein. For example, a derivative of a Tetrahymena L-19 IVS RNA can beconstructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in aTSCX-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; andCech et al. U.S. Pat. No. 5,116,742. Alternatively, TSCX mRNA can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science261:1411-1418.

Alternatively, TSCX gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of a TSCXnucleic acid (e.g., the TSCX promoter and/or enhancers) to form triplehelical structures that prevent transcription of the TSCX gene in targetcells, See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84;Helene. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992)Bioassays 14: 807-15.

In various embodiments, the nucleic acids of TSCX can be modified at thebase moiety, sugar moiety or phosphate backbone to improve, e.g., thestability, hybridization, or solubility of the molecule. For example,the deoxyribose phosphate backbone of the nucleic acids can be modifiedto generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg MedChem 4: 5-23). As used herein, the terms “peptide nucleic acids” or“PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which thedeoxyribose phosphate backbone is replaced by a pseudopeptide backboneand only the four natural nucleobases are retained. The neutral backboneof PNAs has been shown to allow for specific hybridization to DNA andRNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup et al. (1996) above; Perry-O'Keefe etal. (1996) PNAS 93: 14670-675.

PNAs of TSCX can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs ofTSCX can also be used, e.g., in the analysis of single base pairmutations in a gene by, e.g., PNA directed PCR clamping; as artificialrestriction enzymes when used in combination with other enzymes, e.g.,51 nucleases (Hyrup B. (1996) above); or as probes or primers for DNAsequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe(1996), above).

In another embodiment, PNAs of TSCX can be modified, e.g., to enhancetheir stability or cellular uptake, by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of TSCX can be generated that may combinethe advantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNase H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup (1996) above). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. Forexample, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry, and modified nucleosideanalogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used between the PNA and the 5′ end of DNA (Maget al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupledin a stepwise manner to produce a chimeric molecule with a 5′ PNAsegment and a 3′ DNA segment (Finn et al. (1996) above). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652;PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g.,PCT Publication No. WO89/10134). In addition, oligonucleotides can bemodified with hybridization triggered cleavage agents (See, e.g., Krolet al., 1988, BioTechniques 6:958-976) or intercalating agents. (See,e.g., Zon, 1988, Pharm. Res. 5: 539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,a hybridization triggered cross-linking agent, a transport agent, ahybridization-triggered cleavage agent, etc.

TSCX Polypeptides

One aspect of the invention pertains to isolated TSCX proteins, andbiologically active portions thereof, or derivatives, fragments, analogsor homologs thereof. Also provided are polypeptide fragments suitablefor use as immunogens to raise anti-TSCX antibodies. In one embodiment,native TSCX proteins can be isolated from cells or tissue sources by anappropriate purification scheme using standard protein purificationtechniques. In another embodiment, TSCX proteins are produced byrecombinant DNA techniques. Alternative to recombinant expression, aTSCX protein or polypeptide can be synthesized chemically using standardpeptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theTSCX protein is derived, or substantially free from chemical precursorsor other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of TSCXprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of TSCX protein having less than about 30% (by dryweight) of non-TSCX protein (also referred to herein as a “contaminatingprotein”), more preferably less than about 20% of non-TSCX protein,still more preferably less than about 10% of non-TSCX protein, and mostpreferably less than about 5% non-TSCX protein. When the TSCX protein orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of TSCX protein in which the protein isseparated from chemical precursors or other chemicals that are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of TSCX protein having less than about 30% (by dry weight)of chemical precursors or non-TSCX chemicals, more preferably less thanabout 20% chemical precursors or non-TSCX chemicals, still morepreferably less than about 10% chemical precursors or non-TSCXchemicals, and most preferably less than about 5% chemical precursors ornon-TSCX chemicals.

Biologically active portions of a TSCX protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the TSCX protein, e.g., the amino acidsequence encoded by a nucleic acid comprising TSCX 1-20 that includefewer amino acids than the full length TSCX proteins, and exhibit atleast one activity of a TSCX protein. Typically, biologically activeportions comprise a domain or motif with at least one activity of theTSCX protein. A biologically active portion of a TSCX protein can be apolypeptide which is, for example, 10, 25, 50, 100 or more amino acidsin length.

A biologically active portion of a TSCX protein of the present inventionmay contain at least one of the above-identified domains conservedbetween the TSCX proteins. An alternative biologically active portion ofa TSCX protein may contain at least two of the above-identified domains.Another biologically active portion of a TSCX protein may contain atleast three of the above-identified domains. Yet another biologicallyactive portion of a TSCX protein of the present invention may contain atleast four of the above-identified domains.

Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a native TSCXprotein.

In some embodiments, the TSCX protein is substantially homologous to oneof these TSCX proteins and retains its the functional activity, yetdiffers in amino acid sequence due to natural allelic variation ormutagenesis, as described in detail below.

In specific embodiments, the invention includes an isolated polypeptidecomprising an amino acid sequence that is 80% or more identical to thesequence of a polypeptide whose expression is modulated in a mammal towhich TSCXic agent is administered.

Determining Homology Between Two or More Sequences

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are homologous at that position(i.e., as used herein amino acid or nucleic acid “homology” isequivalent to amino acid or nucleic acid “identity”).

The nucleic acid sequence homology may be determined as the degree ofidentity between two sequences. The homology may be determined usingcomputer programs known in the art, such as GAP software provided in theGCG program package. See Needleman and Wunsch 1970 J Mol Biol 48:443-453. Using GCG GAP software with the following settings for nucleicacid sequence comparison: GAP creation penalty of 5.0 and GAP extensionpenalty of 0.3, the coding region of the analogous nucleic acidsequences referred to above exhibits a degree of identity preferably ofat least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS(encoding) part of a DNA sequence comprising TSCX::1-7, 10-13, 19-34,45-53, 58-85, 111-113, 120, 130, 132-134 and 138.

The term “sequence identity” refers to the degree to which twopolynucleotide or polypeptide sequences are identical on aresidue-by-residue basis over a particular region of comparison. Theterm “percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over that region of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I, in the case of nucleic acids) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the region ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. The term “substantialidentity” as used herein denotes a characteristic of a polynucleotidesequence, wherein the polynucleotide comprises a sequence that has atleast 80 percent sequence identity, preferably at least 85 percentidentity and often 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison region.

Chimeric and Fusion Proteins

The invention also provides TSCX chimeric or fusion proteins. As usedherein, an TSCX “chimeric protein” or “fusion protein” comprises an TSCXpolypeptide operatively linked to a non-TSCX polypeptide. A “TSCXpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to TSCX, whereas a “non-TSCX polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinthat is not substantially homologous to the TSCX protein, e.g., aprotein that is different from the TSCX protein and that is derived fromthe same or a different organism. Within an TSCX fusion protein the TSCXpolypeptide can correspond to all or a portion of an TSCX protein. Inone embodiment, an TSCX fusion protein comprises at least onebiologically active portion of an TSCX protein. In another embodiment,an TSCX fusion protein comprises at least two biologically activeportions of an TSCX protein. In yet another embodiment, an TSCX fusionprotein comprises at least three biologically active portions of an TSCXprotein. Within the fusion protein, the term “operatively linked” isintended to indicate that the TSCX polypeptide and the non-TSCXpolypeptide are fused in-frame to each other. The non-TSCX polypeptidecan be fused to the N-terminus or C-terminus of the TSCX polypeptide.

For example, in one embodiment an TSCX fusion protein comprises an TSCXdomain operably linked to the extracellular domain of a second protein.Such fusion proteins can be further utilized in screening assays forcompounds which modulate TSCX activity (such assays are described indetail below).

In yet another embodiment, the fusion protein is a GST-TSCX fusionprotein in which the TSCX sequences are fused to the C-terminus of theGST (i.e., glutathione S-transferase) sequences. Such fusion proteinscan facilitate the purification of recombinant TSCX.

In another embodiment, the fusion protein is an TSCX protein containinga heterologous signal sequence at its N-terminus. For example, a nativeTSCX signal sequence can be removed and replaced with a signal sequencefrom another protein. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of TSCX can be increased through useof a heterologous signal sequence.

In yet another embodiment, the fusion protein is an TSCX-immunoglobulinfusion protein in which the TSCX sequences comprising one or moredomains are fused to sequences derived from a member of theimmunoglobulin protein family. The TSCX-immunoglobulin fusion proteinsof the invention can be incorporated into pharmaceutical compositionsand administered to a subject to inhibit an interaction between a TSCXligand and a TSCX protein on the surface of a cell, to thereby suppressTSCX-mediated signal transduction in vivo. The TSCX-immunoglobulinfusion proteins can be used to affect the bioavailability of an TSCXcognate ligand. Inhibition of the TSCX ligand/TSCX interaction may beuseful therapeutically for both the treatments of proliferative anddifferentiative disorders, as well as modulating (e.g. promoting orinhibiting) cell survival. Moreover, the TSCX-immunoglobulin fusionproteins of the invention can be used as immunogens to produce anti-TSCXantibodies in a subject, to purify TSCX ligands, and in screening assaysto identify molecules that inhibit the interaction of TSCX with a TSCXligand.

An TSCX chimeric or fusion protein of the invention can be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). An TSCX-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to theTSCX protein.

TSCX Agonists and Antagonists

The present invention also pertains to variants of the TSCX proteinsthat function as either TSCX agonists (mimetics) or as TSCX antagonists.Variants of the TSCX protein can be generated by mutagenesis, e.g.,discrete point mutation or truncation of the TSCX protein. An agonist ofthe TSCX protein can retain substantially the same, or a subset of, thebiological activities of the naturally occurring form of the TSCXprotein. An antagonist of the TSCX protein can inhibit one or more ofthe activities of the naturally occurring form of the TSCX protein by,for example, competitively binding to a downstream or upstream member ofa cellular signaling cascade which includes the TSCX protein. Thus,specific biological effects can be elicited by treatment with a variantof limited function. In one embodiment, treatment of a subject with avariant having a subset of the biological activities of the naturallyoccurring form of the protein has fewer side effects in a subjectrelative to treatment with the naturally occurring form of the TSCXproteins.

Variants of the TSCX protein that function as either TSCX agonists(mimetics) or as TSCX antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theTSCX protein for TSCX protein agonist or antagonist activity. In oneembodiment, a variegated library of TSCX variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of TSCX variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential TSCX sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of TSCX sequences therein. There are avariety of methods which can be used to produce libraries of potentialTSCX variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential TSCX sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323; Itakuraet al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 11:477.

Polypeptide Libraries

In addition, libraries of fragments of the TSCX protein coding sequencecan be used to generate a variegated population of TSCX fragments forscreening and subsequent selection of variants of an TSCX protein. Inone embodiment, a library of coding sequence fragments can be generatedby treating a double stranded PCR fragment of a TSCX coding sequencewith a nuclease under conditions wherein nicking occurs only about onceper molecule, denaturing the double stranded DNA, renaturing the DNA toform double stranded DNA that can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal and internalfragments of various sizes of the TSCX protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of TSCX proteins. The mostwidely used techniques, which are amenable to high throughput analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique that enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify TSCX variants (Arkin and Yourvan (1992) PNAS 89:7811-7815;Delgrave et al. (1993) Protein Engineering 6:327-331).

Anti-TSCX Antibodies

An isolated TSCX protein, or a portion or fragment thereof, can be usedas an immunogen to generate antibodies that bind TSCX using standardtechniques for polyclonal and monoclonal antibody preparation. Thefull-length TSCX protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of TSCX for use as immunogens. Theantigenic peptide of TSCX comprises at least 8 amino acid residues ofthe amino acid sequence encoded by a nucleic acid comprising the nucleicacid sequence shown in TSC: 1-8, 10-12, and 15-25 and encompasses anepitope of TSCX such that an antibody raised against the peptide forms aspecific immune complex with TSCX. Preferably, the antigenic peptidecomprises at least 10 amino acid residues, more preferably at least 15amino acid residues, even more preferably at least 20 amino acidresidues, and most preferably at least 30 amino acid residues. Preferredepitopes encompassed by the antigenic peptide are regions of TSCX thatare located on the surface of the protein, e.g., hydrophilic regions. Asa means for targeting antibody production, hydropathy plots showingregions of hydrophilicity and hydrophobicity may be generated by anymethod well known in the art, including, for example, the Kyte Doolittleor the Hopp Woods methods, either with or without Fouriertransformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci.USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142,each incorporated herein by reference in their entirety.

TSCX polypeptides or derivatives, fragments, analogs or homologsthereof, may be utilized as immunogens in the generation of antibodiesthat immunospecifically-bind these protein components. The term“antibody” as used herein refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that specifically binds(immunoreacts with) an antigen. Such antibodies include, but are notlimited to, polyclonal, monoclonal, chimeric, single chain, F_(ab) andF_((ab′)2) fragments, and an F_(ab) expression library. Variousprocedures known within the art may be used for the production ofpolyclonal or monoclonal antibodies to an TSCX protein sequence, orderivatives, fragments, analogs or homologs thereof. Some of theseproteins are discussed below.

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byinjection with the native protein, or a synthetic variant thereof, or aderivative of the foregoing. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed TSCX protein or achemically synthesized TSCX polypeptide. The preparation can furtherinclude an adjuvant. Various adjuvants used to increase theimmunological response include, but are not limited to, Freund's(complete and incomplete), mineral gels (e.g., aluminum hydroxide),surface active substances (e.g., lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, dinitrophenol, etc.), humanadjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, orsimilar immunostimulatory agents. If desired, the antibody moleculesdirected against TSCX can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction.

The term “monoclonal antibody” or “monoclonal antibody composition”, asused herein, refers to a population of antibody molecules that containonly one species of an antigen binding site capable of immunoreactingwith a particular epitope of TSCX. A monoclonal antibody compositionthus typically displays a single binding affinity for a particular TSCXprotein with which it immunoreacts. For preparation of monoclonalantibodies directed towards a particular TSCX protein, or derivatives,fragments, analogs or homologs thereof, any technique that provides forthe production of antibody molecules by continuous cell line culture maybe utilized. Such techniques include, but are not limited to, thehybridoma technique (see Kohler & Milstein, 1975 Nature 256: 495-497);the trioma technique; the human B-cell hybridoma technique (see Kozbor,et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique toproduce human monoclonal antibodies (see Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies may be utilized in the practice ofthe present invention and may be produced by using human hybridomas (seeCote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or bytransforming human B-cells with Epstein Barr Virus in vitro (see Cole,et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,Inc., pp. 77-96).

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to a TSCX protein (sec e.g., U.S.Pat. No. 4,946,778). In addition, methods can be adapted for theconstruction of F_(ab) expression libraries (see e.g., Huse, et al.,1989 Science 246: 1275-1281) to allow rapid and effective identificationof monoclonal F_(ab) fragments with the desired specificity for a TSCXprotein or derivatives, fragments, analogs or homologs thereof.Non-human antibodies can be “humanized” by techniques well known in theart. See e.g., U.S. Pat. No. 5,225,539. Antibody fragments that containthe idiotypes to a TSCX protein may be produced by techniques known inthe art including, but not limited to: (i) an F_((ab′)2) fragmentproduced by pepsin digestion of an antibody molecule; (ii) an F_(ab)fragment generated by reducing the disulfide bridges of an F_((ab′)2)fragment; (iii) an F_(ab) fragment generated by the treatment of theantibody molecule with papain and a reducing agent and (iv) F_(\),fragments.

Additionally, recombinant anti-TSCX antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCTInternational Application No. PCT/US86/02269; European PatentApplication No. 184,187; European Patent Application No. 171,496;European Patent Application No. 173,494; PCT International PublicationNo. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent ApplicationNo. 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.(1987) PNAS 84:3439-3443; Liu et al. (1987) J Immunol. 139:3521-3526;Sun et al. (1987) PNAS 84:214-218; Nishimura et al. (1987) Cancer Res47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) JNatl Cancer Inst. 80:1553-1559); Morrison (1985) Science 229:1202-1207;Oi et al. (1986) BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones etal. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534;and Beidler et al. (1988) J Immunol 141:4053-4060.

In one embodiment, methods for the screening of antibodies that possessthe desired specificity include, but are not limited to, enzyme-linkedimmunosorbent assay (ELISA) and other immunologically-mediatedtechniques known within the art. In a specific embodiment, selection ofantibodies that are specific to a particular domain of a TSCX protein isfacilitated by generation of hybridomas that bind to the fragment of aTSCX protein possessing such a domain. Antibodies that are specific forone or more domains within a TSCX protein, e.g., domains spanning theabove-identified conserved regions of TSCX family proteins, orderivatives, fragments, analogs or homologs thereof, are also providedherein.

Anti-TSCX antibodies may be used in methods known within the artrelating to the localization and/or quantitation of a TSCX protein(e.g., for use in measuring levels of the TSCX protein withinappropriate physiological samples, for use in diagnostic methods, foruse in imaging the protein, and the like). In a given embodiment,antibodies for TSCX proteins, or derivatives, fragments, analogs orhomologs thereof, that contain the antibody derived binding domain, areutilized as pharmacologically-active compounds [hereinafter“Therapeutics”].

An anti-TSCX antibody (e.g., monoclonal antibody) can be used to isolateTSCX by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-TSCX antibody can facilitate thepurification of natural TSCX from cells and of recombinantly producedTSCX expressed in host cells. Moreover, an anti-TSCX antibody can beused to detect TSCX protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the TSCX protein. Anti-TSCX antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phyeoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

TSCX Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding TSCX protein, orderivatives, fragments, analogs or homologs thereof. As used herein, theterm “vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a linear or circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODSIN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those that direct constitutive expression of anucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., TSCX proteins, mutant forms ofTSCX, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of TSCX in prokaryotic or eukaryotic cells. For example, TSCXcan be expressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors) yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: (1) to increase expression ofrecombinant protein; (2) to increase the solubility of the recombinantprotein; and (3) to aid in the purification of the recombinant proteinby acting as a ligand in affinity purification. Often, in fusionexpression vectors, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the recombinant protein to enableseparation of the recombinant protein from the fusion moiety subsequentto purification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, Gottesman, GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 119-128, Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:211:1-7, 10-13, 19-34, 45-53, 58-85, 111-113, 120, 130, 132-134and 13518). Such alteration of nucleic acid sequences of the inventioncan be carried out by standard DNA synthesis techniques.

In another embodiment, the TSCX expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al., (1987) EMBO J 6:229-234), pMFa(Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al.,(1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, TSCX can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., SF9 cells) include the pAcseries (Smith et al. (1983) Mol Cell Biol 3:2156-2165) and the pVLseries (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 ofSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv Immunol 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, e.g., themurine hox promoters (Kessel and Gruss (1990) Science 249:374-379) andthe α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to TSCX mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosen thatdirect the continuous expression of the antisense RNA molecule in avariety of cell types, for instance viral promoters and/or enhancers, orregulatory sequences can be chosen that direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub et al., “Antisense RNA asa molecular tool for genetic analysis,” Reviews—Trends in Genetics, Vol.1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, TSCXprotein can be expressed in bacterial cells such as E. coli, insectcells, yeast or mammalian cells (such as Chinese hamster ovary cells(CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MOLECULARCLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding TSCX or can be introduced on a separate vector. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) an TSCX protein.Accordingly, the invention further provides methods for producing TSCXprotein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding TSCX has been introduced) in asuitable medium such that TSCX protein is produced. In anotherembodiment, the method further comprises isolating TSCX from the mediumor the host cell.

Kits and Nucleic Acid Collections for Identifying TSCX Nucleic Acids

In another aspect, the invention provides a kit useful for examiningTSCXicity of agents. The kit can include nucleic acids that detect twoor more TSCX sequences. In preferred embodiments, the kit includesreagents which detect 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 50, 100 or allof the TSCX nucleic acid sequences.

The invention also includes an isolated plurality of sequences which canidentify one or more TSCX responsive nucleic acid sequences.

The kit or plurality may include, e.g., sequence homologous to TSCXnucleic acid sequences, or sequences which can specifically identify oneor more TSCX nucleic acid sequences.

EXAMPLES Example 1 Expression Analysis of Antileukoprotease in VariousTissues

The quantitative expression of NMB (GenBank™ Accession No: AJ251685;Table 1; NMB or TSC122, listed in Table 1 on page 10) was assessed usingmicrotiter plates containing RNA samples from a variety of normal andpathology-derived cells, cell lines and tissues using real timequantitative PCR(RTQ PCR; TAQMAN®). RTQ PCR was performed on aPerkin-Elmer Biosystems ABI PRISM® 7700 Sequence Detection System.Various collections of samples are assembled on the plates, and referredto as Panel 1 (containing cells and cell lines from normal and cancersources), and Panel 2 (containing samples derived from tissues, inparticular from surgical samples, from normal and cancer sources). Theamino acid sequence of the NMB polypeptide is

(SEQ ID NO: 27) MESLCGVLGFLLLAAGLPLQAAKRFRDVLGHEQYPDHMREHNQLRGWSSDENEWDEHLYPVWRRGDGRWKDSWEGGRVQAVLTSDSPALVGSNITFVVNLVFRCQKEDANGNIVYEKNCRNDLGLTSDLHVYNWTAGADDGDWEDGTSRSQHLRFPDRRPFPRPHGWKKWSFVYVFHTLGQYFQKLGRCSARVSINTVNLTAGPQVMEVTVFRRYGRAYIPISKVKDVYVITDQIPVFVTMSQKNDRNLSDEIFLRDLPIVFDVLIHDPSHFLNDSAISYKWNFGDNTGLFVSNNHTLNHTYVLNGTFNLNLTVQTAVPGPCPPPSPSTPPSPSTPPLPSPSPLPTLSTPSPSLMPTGYKSMELSDISNENCRINRYGYFRATITIVEGILEVSIMQIADVPMPTPQPANSLMDFTVTCKGATPMEACTIISDPTCQIAQNRVCSPVAVDGLCLLSVRRAFNGSGTYCVNFTLGDDASLALTSTLISIPGKDPDSPLRAVNGVLISIGCLAVLVTMVTILLYKKHKAYKPIGNCPRNTVKGKGLSVLLSHAKAPFFRGDQEKDPLLQDKPRTL.The nucleotide sequence encoding this protein is

(GenBank ™ Accession No: AJ251685) (SEQ ID NO: 26)atcgcaggaggccgacactgtgactcctggtggatcgggactggggagtcagagtcaagccctgactggttgcaggcgctcggagtcagcatggaaagtctctgcggggtcctgggatttctgctgctggctgcaggactgcctctccaggctgccaagcgatttcgtgatgtgctgggccatgaacagtatcccgatcacatgagagagcacaaccaattacgtggctggtcttcggatgaaaatgaatgggatgaacacctgtatccagtgtggaggaggggagacggcaggtggaaggactcctgggaaggaggccgtgtgcaggcagtcctgaccagtgactcaccggctctggtgggttccaatatcacttttgtggtgaacctggtgttccccagatgccagaaggaagatgctaatggcaatatcgtctatgagaagaactgcaggaatgatttgggactgacatctgacctgcatgtctacaactggactgcaggggcagatgatggtgactgggaagatggcaccagccgaagccagcatctcaggttcccggacaggaggcccttccctcgcccccatggatggaagaaatggagctttgtctacgtctttcacacacttggccagtatttccaaaaactgggtcggtgttcagcacgggtttctataaacacagtcaacttgacagctggccctcaggtcatggaagtgactgtctttcgaagatacggccgggcatacattcccatctcgaaggtgaaagatgtgtatgtgataacagatcagatccctgtattcgtgaccatgtcccagaagaatgacaggaacttgtctgatgagatcttcctcagagacctccccatcgtcttcgatgtcctcattcatgatcccagccacttcctcaacgactctgccatttcctacaagtggaactttggggacaacactggcctgtttgtctccaacaatcacactttgaatcacacttatgtgctcaatggaaccttcaaccttaacctcaccgtgcaaactgcagtgcccgggccatgccctcccccttcgccttcgactccgccttcaccttcaactccgcccttaccttcgccctcacctttgcccacattatcaacacctagcccctctttaatgcctactggttacaaatccatggagctgagtgacatttccaatgaaaactgccgaataaacagatatggctacttcagagccaccatcacaattgtagaggggatcctggaagtcagcatcatgcagatagcagatgtccccatgcccacaccgcagcctgccaactccctgatggacttcactgtgacctgcaaaggggccacccccatggaagcctgtacgatcatctccgaccccacctgccagatcgcccagaaccgggtctgcagccctgtggctgtggatgggctgtgcctgctgtctgtgagaagagccttcaatgggtctggcacctactgtgtgaatttcactctgggagatgatgcaagcctggccctcaccagcaccctgatctctatccctggcaaagacccagactcccctctgagagcagtgaatggtgtcctgatctccatcggctgcctggctgtgcttgtcaccatggttaccatcttgctgtacaaaaaacacaaggcgtacaagccaataggaaactgccccaggaacacggtcaagggcaagggcctgagtgactcctcagtcacgcgaaagccccgttcttccgaggagaccaggagaaggatccattgctccaggacaagccaaggacactctaagtctttggccttccctctgaccaggaacccactcttctgtgcatgtatgtgagctgtgcagaagtatgtggctgggaactgagttctctaaggattattgtaaaatgtatatcgtggcttagggagtgtggttaaatagcattttagagaagacatgggaagacttagtgtttcttcccatctgtattgtggtttttacactgttcgtggggtggacacgctgtgtctgaaggggaggtggggtcactgctacttaaggtcctaggttaactgggggagataccacagatgccttcagctttccacataacatgggcatgaacccagctaatcaccacctgaaggccatgcttcatctgccttccaactcactgagcatgcctgagctcctgacaaaattataatgggcccgggcttttgtgtatgggtgccgtgtggtgtacatattctactcattaaaaaaggcagtctaaaaaaaaaaaaaaa aaa.

First, the RNA samples were normalized to constitutively expressed genessuch as β-actin and GAPDH. RNA (˜50 ng total or ˜1 ng polyA+) wasconverted to cDNA using the TAQMAN® Reverse Transcription Reagents Kit(PE Biosystems, Foster City, Calif.; Catalog No. N808-0234) and randomhexamers according to the manufacturer's protocol. Reactions wereperformed in 20 ul and incubated for 30 min. at 48° C. cDNA (5 ul) wasthen transferred to a separate plate for the TAQMAN® reaction usingβ-actin and GAPDH TAQMAN® Assay Reagents (PE Biosystems; Catalog Nos.4310881E and 4310884E, respectively) and TAQMAN® universal PCR MasterMix (PE Biosystems; Catalog No. 4304447) according to the manufacturer'sprotocol. Reactions were performed in 25 ul using the followingparameters: 2 min. at 50° C.; 10 min. at 95° C.; 15 sec. at 95° C./1min. at 60° C. (40 cycles). Results were recorded as CT values (cycle atwhich a given sample crosses a threshold level of fluorescence) using alog scale, with the difference in RNA concentration between a givensample and the sample with the lowest CT value being represented as 2 tothe power of delta CT. The percent relative expression is then obtainedby taking the reciprocal of this RNA difference and multiplying by 100.The average CT values obtained for β-actin and GAPDH were used tonormalize RNA samples. The RNA sample generating the highest CT valuerequired no further diluting, while all other samples were dilutedrelative to this sample according to their β-actin/GAPDH average CTvalues.

Normalized RNA (5 ul) was converted to cDNA and analyzed via TAQMAN®using One Step RT-PCR Master Mix Reagents (PE Biosystems; Catalog No.4309169) and gene-specific primers according to the manufacturer'sinstructions. Probes and primers were designed for each assay accordingto Perkin Elmer Biosystem's Primer Express Software package (version Ifor Apple Computer's Macintosh Power PC) or a similar algorithm usingthe target sequence as input. Default settings were used for reactionconditions and the following parameters were set before selectingprimers: primer concentration=250 nM, primer melting temperature (T_(m))range=58°-60° C., primer optimal Tm=59° C., maximum primer difference=2°C., probe does not have 5′ G, probe T_(m) must be 10° C. greater thanprimer T_(m), amplicon size 75 bp to 100 bp. The probes and primersselected (see below) were synthesized by Synthegen (Houston, Tex., USA).Probes were double purified by HPLC to remove uncoupled dye andevaluated by mass spectroscopy to verify coupling of reporter andquencher dyes to the 5′ and 3′ ends of the probe, respectively. Theirfinal concentrations were: forward and reverse primers, 900 nM each, andprobe, 200 nM.

PCR conditions: Normalized RNA from each tissue and each cell line wasspotted in each well of a 96 well PCR plate (Perkin Elmer Biosystems).PCR cocktails including two probes (a probe specific for the targetclone and another gene-specific probe multiplexed with the target probe)were set up using 1× TaqMan™ PCR Master Mix for the PE Biosystems 7700,with 5 mM MgCl2, dNTPs (dA, G, C, U at 1:1:1:2 ratios), 0.25 U/mlAmpliTaq Gold™ (PE Biosystems), and 0.4 U/μl RNase inhibitor, and 0.25U/μl reverse transcriptase. Reverse transcription was performed at 48°C. for 30 minutes followed by amplification/PCR cycles as follows: 95°C. 10 min, then 40 cycles of 95° C. for 15 seconds, 60° C. for 1 minute.

In the results for Panel 1, the following abbreviations are used:

ca.=carcinoma,*=established from metastasis,met=metastasis,s cell var=small cell variant,non-s=non-sm=non-small,squam=squamous,pl. eff=pl effusion=pleural effusion,glio=glioma,astro=astrocytoma, andneuro=neuroblastoma.

Panel 2

The plates for Panel 2 generally include 2 control wells and 94 testsamples composed of RNA or cDNA isolated from human tissue procured bysurgeons working in close cooperation with the National CancerInstitute's Cooperative Human Tissue Network (CHTN) or the NationalDisease Research Initiative (NDRI). The tissues are derived from humanmalignancies and in cases where indicated many malignant tissues have“matched margins” obtained from noncancerous tissue just adjacent to thetumor. These are termed normal adjacent tissues and are denoted “NAT” inthe results below. The tumor tissue and the “matched margins” areevaluated by two independent pathologists (the surgical pathologists andagain by a pathologists at NDRI or CHTN). This analysis provides a grosshistopathological assessment of tumor differentiation grade. Moreover,most samples include the original surgical pathology report thatprovides information regarding the clinical stage of the patient. Thesematched margins are taken from the tissue surrounding (i.e. immediatelyproximal) to the zone of surgery (designated “NAT”, for normal adjacenttissue, in Table 4). In addition, RNA and cDNA samples were obtainedfrom various human tissues derived from autopsies performed on elderlypeople or sudden death victims (accidents, etc.). These tissue wereascertained to be free of disease and were purchased from variouscommercial sources such as Clontech (Palo Alto, Calif.), ResearchGenetics, and Invitrogen.

RNA integrity from all samples is controlled for quality by visualassessment of agarose gel electropherograms using 28S and 18S ribosomalRNA staining intensity ratio as a guide (2:1 to 2.5:1 28s:18s) and theabsence of low molecular weight RNAs that would be indicative ofdegradation products. Samples are controlled against genomic DNAcontamination by RTQ PCR reactions run in the absence of reversetranscriptase using probe and primer sets designed to amplify across thespan of a single exon.

The TaqMan™ expression profiles of NMB were generated using a specificgene probes and primer set (Ag 817) as shown below:

Ag 817 forward: [SEQ ID NO.: 23] 5′-TCAATGGAACCTTCAGCCTTA-3′ ProbeTET:[SEQ ID NO.: 24] 5′-CTCACTGTGAAAGCTGCAGCACCAG-3′-TAMRA Reverse: [SEQ IDNO.: 25] 5′-GAAGGGGTGGGTTTTGAAG-3′.

The results shown in Table 2 (see below) relate to 41 normal humantissues and 55 human cancer cell lines and demonstrate the highexpression of NMB in melanomas cell lines and overexpression in thebreast cancer cell line MDA-N. The results shown in Table 3 (see below)relate to additional tumor tissues, many of which are matched withnormal adjacent tissue (NAT), as defined by the operating surgeon thatobtained the samples. It reveals that NMB is overexpressed in 9/9 kidneytumors compared either with normal kidney or NAT. This analysiscorroborates the GeneCalling™ results which originally identified theexpression of NMB that NMB is also overexpressed in some of the lungcarcinoma tissues compared with NATs and 2 melanoma metastasis comparedwith NAT.

NCI's CGAP Sage analysis indicates that NMB is expressed in severalglioblastoma (H392, pooled GBM, GBMH1110), and in 1 malignant breasttumor (SKBR3), in accordance with panel 1 TaqMan analysis. NCI data forEST expression, called “body map”, reveals that NMB is expressed inSchwann cells, in adenocarcinoma and s.cell carcinoma.

Based on NMB's gene expression profile and its homology with pMEL17, itis anticipated that for a subset of human tumors including renal cellcarcinomas, lung carcinomas, melanomas and CNS cancers, successfultargeting of NMB using a monoclonal antibody will have an inhibitoryeffect on tumor growth, matrix invasion and metastatic dissemination.Furthermore, targeting of NMB will have a therapeutic effect on the TSCdisease.

Furthermore, in consideration of NMB potential enzymatic activity, NMBcould be used as a target for screening a small molecule drug.

In summary, these results demonstate the relevance of NMB as atherapeutic target for the treatment of TSC is strengthened by itsexpression/overexpression in several tissues that are affected in TSC.

TABLE 2 Taq Man results for PANEL 1 Rel. Expr., % Tissue Name1.2tm958t_ag817 Endothelial cells 0 Heart (fetal) 5.4 Pancreas 6Pancreatic ca. CAPAN 2 0 Adrenal Gland (new lot*) 2.7 Thyroid 19.3Salavary gland 2.7 Pituitary gland 3.7 Brain (fetal) 0.8 Brain (whole)2.4 Brain (amygdala) 1.6 Brain (cerebellum) 0.4 Brain (hippocampus) 1.3Brain (thalamus) 1.1 Cerebral Cortex 1.2 Spinal cord 7.6 CNS ca.(glio/astro) U87-MG 27,2 CNS ca. (glio/astro) U-118-MG 13.5 CNS ca.(astro) SW1783 0.4 CNS ca.* (neuro; met) SK-N-AS 0.7 CNS ca. (astro)SF-539 52.9 CNS ca. (astro) SNB-75 7 CNS ca. (glio) SNB-19 1.3 CNS ca.(glio) U251 4.9 CNS ca. (glio) SF-295 11 Heart 17.1 Skeletal Muscle (newlot*) 5.7 Bone marrow 0.8 Thymus 9.9 Spleen 5 Lymph node 25.7 Colorectal8.2 Stomach 5.6 Small intestine 8.1 Colon ca. SW480 0 Colon ca.* (SW480met)SW620 0 Colon ca. HT29 0 Colon ca. HCT-116 0 Colon ca. CaCo-2 083219 CC Well to Mod Diff 2.4 (ODO3866) Colon ca. HCC-2998 0.1 Gastricca.* (liver met) NCI-N87 18.2 Bladder 8.1 Trachea 7.4 Kidney 3.1 Kidney(fetal) 1.7 Renal ca. 786-0 0 Renal ca. A498 4.7 Renal ca. RXF 393 1.5Renal ca. ACHN 0 Renal ca. UO-31 1.8 Renal ca. TK-10 0 Liver 2.5 Liver(fetal) 2.3 Liver ca. (hepatoblast) HepG2 0 Lung 21

TABLE 3 Taq Man Results for Panel 2 Rel. Expr., % Tissue Name2tm1063t_ag817 Normal Colon GENPAK 061003 11.8 83219 CC Well to Mod Diff(ODO3866) 0 83220 CC NAT (ODO3866) 9.1 83221 CC Gr. 2 rectosigmoid(ODO3868) 1.4 83222 CC NAT (ODO3868) 7.1 83235 CC Mod Diff (OD03920) 1.283236 CC NAT (ODO3920) 1.2 83237 CC Gr. 2 ascend colon (ODO3921) 4.883238 CC NAT (ODO3921) 5.8 83241 CC from Partial Hepatectomy (ODO4309)7.8 83242 Liver NAT (ODO4309) 2.9 87472 Colon mets to lung (OD04451-01)14.6 87473 Lung NAT (OD04451-02) 19.8 Normal Prostate Clontech A+ 6546-18.8 84140 Prostate Cancer (OD04410) 2.9 84141 Prostate NAT (OD04410) 0.787073 Prostate Cancer (OD04720-01) 1 87074 Prostate NAT (OD04720-02) 1.5Normal Lung GENPAK 061010 49.3 83239 Lung Met to Muscle (ODO4286) 74.783240 Muscle NAT (ODO4286) 6.5 84136 Lung Malignant Cancer (OD03126)10.4 84137 Lung NAT (OD03126) 4.6 84871 Lung Cancer (OD04404) 27.7 84872Lung NAT (OD04404) 7.9 84875 Lung Cancer (OD04565) 41.8 84876 Lung NAT(OD04565)** 3.8 85950 Lung Cancer (OD04237-01) 10.1 85970 Lung NAT(OD04237-02) 1.5 83255 Ocular Mel Met to Liver (ODO4310) 77.4 83256Liver NAT (ODO4310) 1.8 84139 Melanoma Mets to Lung (OD04321) 53.6 84138Lung NAT (OD04321) 5.8 Normal Kidney GENPAK 061008 10.1 83786 Kidney Ca,Nuclear grade 2 (OD04338) 22.5 83787 Kidney NAT (OD04338) 1.3 83788Kidney Ca Nuclear grade 1/2 (OD04339) 17.2 83789 Kidney NAT (OD04339) 283790 Kidney Ca, Clear cell type (OD04340) 11.3 83791 Kidney NAT(OD04340) 3.7 83792 Kidney Ca, Nuclear grade 3 (OD04348) 12.1 83793Kidney NAT (OD04348) 1.9 87474 Kidney Cancer (OD04622-01) 19.6 87475Kidney NAT (OD04622-03) 9 85973 Kidney Cancer (OD04450-01) 54.7 85974Kidney NAT (OD04450-03) 2.7 Kidney Cancer Clontech 8120613 67.8 KidneyNAT Clontech 8120614 5.8 Kidney Cancer Clontech 9010320 56.3 Kidney NATClontech 9010321 7.2 Kidney Cancer Clontech 8120607 100 Kidney NATClontech 8120608 10.2 Normal Uterus GENPAK 061018 11.5 Uterus CancerGENPAK 064011 2 Normal Thyroid Clontech A+ 6570-1** 44.4 Thyroid CancerGENPAK 064010 90.1 Thyroid Cancer INVITROGEN A302152 10.9 Thyroid NATINVITROGEN A302153 8.3 Normal Breast GENPAK 061019 2.4 84877 BreastCancer (OD04566) 5.5 Breast Cancer Res. Gen. 1024 7.1 85975 BreastCancer (OD04590-01) 1.7 85976 Breast Cancer Mets (OD04590-03) 2 87070Breast Cancer Metastasis (OD04655-05) 1.6 GENPAK Breast Cancer 0640063.4 Breast Cancer Clontech 9100266 11.1 Breast NAT Clontech 9100265 7.7Breast Cancer INVITROGEN A209073 11 Breast NAT INVITROGEN A2090734 3.2Normal Liver GENPAK 061009 6 Liver Cancer Research Genetics RNA 102636.3 Liver Cancer Research Genetics RNA 1025 4 Paired Liver CancerTissue Research Genetics RNA 10.4 6004-T Paired Liver Tissue ResearchGenetics RNA 6004-N 32.1 Paired Liver Cancer Tissue Research GeneticsRNA 44.4 6005-T Paired Liver Tissue Research Genetics RNA 6005-N 40.6Liver Cancer GENPAK 064003 18.4 Normal Bladder GENPAK 061001 19.9Bladder Cancer Research Genetics RNA 1023 17 87071 Bladder Cancer(OD04718-01) 1.4 87072 Bladder Normal Adjacent (OD04718-03) 0.9 BladderCancer INVITROGEN A302173 43.8 Normal Ovary Res. Gen. 39.5 OvarianCancer GENPAK 064008 10.8 87492 Ovary Cancer (OD04768-07) 5 87493 OvaryNAT (OD04768-08) 6.2 Normal Stomach GENPAK 061017 37.4 Gastric CancerClontech 9060358** 7.4 NAT Stomach Clontech 9060359 14.6 Gastric CancerClontech 9060397 40.9 NAT Stomach Clontech 9060396 9.9 Gastric CancerClontech 9060395 20.9 NAT Stomach Clontech 9060394 22.2 Gastric CancerGEN PAK 064005 8.6 genomic DNA control 4.5 Chemistry Control 0.1

Example 2 Therapeutic Targeting of CYR61

Based on CYR61's gene expression profile, it is anticipated that for asubset of human tumors including renal cell carcinomas, lung carcinomas,melanomas and CNS cancers, successful targeting of CYR61 using amonoclonal antibody will have an inhibitory effect on tumor growth,matrix invasion and metastatic dissemination. Furthermore, targeting ofCYR61 will have a therapeutic effect on the TSC disease.

Example 3 Therapeutic Targeting of NET-7

NET-7 is overexpressed by a breast cancer cell lines and it is regulatedby estradiol treatment of a ER positive cell line MCF7. Based on NET-7'sgene expression profile, it is anticipated that for a subset of humantumors specifically breast tumors, successful targeting of NET-7 using amonoclonal antibody will have an inhibitory effect on tumor growth,matrix invasion and metastatic dissemination. Furthermore, targeting ofNET-7 will have a therapeutic effect on the TSC disease adrenomedullinprecursor (and Receptor activity modifying protein 1)

NET-7 has potent and long-lasting vasodilatory effects in severalvascular systems. In addition to adrenomedullin, another hypotensivepeptide, proadrenomedullin-derived peptide (PAMP), was also found to beprocessed from the adrenomedullin precursor. PAMP inhibits neuraltransmission at peripheral sympathetic nerve endings, althoughadrenomedullin directly dilates vascular smooth muscle. Adrenomedullinmight participate in the pathogenesis of hypertension, renal failure andcongestive heart failure. Receptor activity-modifying proteins (RAMPs)are single-transmembrane proteins that transport the calcitoninreceptor-like receptor (CRLR) to the cell surface. RAMP 1-transportedCRLR is a calcitonin gene-related peptide (CGRP) receptor. RAMP1 isdownregulated in NSC. Because of its activities, overexpression ofadrenomedullin precursor by TSC patients might explain some of the TSC

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of diagnosing or determining thesusceptibility to a tuberous sclerosis complex associated disorder in asubject, the method comprising: (a) providing from the subject a testcell population comprising cells capable of expressing one or morenucleic acid sequences selected from the group consisting of TSC 1-8,10-12, 15-141 and 142; (b) measuring expression of one or more of thenucleic acid sequences in the test cell population; and (c) comparingthe expression of the nucleic acid sequences in the test cell populationto the expression of the nucleic acid sequences in a reference profilecomprising at least one cell from a subject not suffering from atuberous sclerosis complex associated disorder; and (d) identifying adifference in expression levels of the nucleic acid sequences, ifpresent, in the test cell population and reference profile, therebydiagnosing or determining the susceptibility to a tuberous sclerosiscomplex associated disorder in the subject.
 2. The method of claim 1,wherein the subject is a human.
 3. The method of claim 1, wherein thetuberous sclerosis complex associated disorder is selected from thegroup consisting of hamartomas, hamartias, renal carcinoma, malignantangiomyolipoma, hypomelanotic macules, facila angiofibroma, shagreenpatches, and ungula fibromas.
 4. The method of claim 1, wherein themethod comprises comparing the expression of five or more of the nucleicacid sequences, comparing the expression of 20 or more of the nucleicacid sequences or comparing the expression of 25 or more of the nucleicacid sequences.
 5. An isolated nucleic acid comprising a nucleic acidsequence selected from the group consisting of a TSC 1-8, 10-12, 15-25gene, and its complement.
 6. A vector comprising the nucleic acid ofclaim
 5. 7. A cell comprising the vector of claim
 6. 8. A polypeptideencoded by the nucleic acid of claim
 5. 9. A method of treating adisease or disorder characterized by aberrant expression or activity ofNMB, said method comprising administering the isolated antibody thatimmunospecifically binds to an NMB polypeptide.
 10. The method of claim9, wherein the antibody inhibits expression or activity of NMB.
 11. Themethod of claim 9, wherein the disease or disorder characterized byaberrant expression or activity of NMB is a cancer.
 12. The method ofclaim 11, wherein the cancer is breast cancer.
 13. The method of claim11, wherein the cancer is selected from the group consisting ofmelanoma, renal carcinoma, lung carcinoma, and a CNS cancer.
 14. Themethod of claim 11, wherein the cancer is a metastasis from a primarytumor.
 15. The method of claim 14, wherein the primary tumor is selectedfrom the group consisting of a breast cancer tumor, a melanoma, a renalcell carcinoma, a lung carcinoma, and a CNS cancer.
 16. The method ofclaim 9, wherein the antibody is administered prior to the manifestationof a symptom associated with aberrant expression or activity of NMB,thereby delaying the progression of the disease or disorder.
 17. Themethod of claim 9, wherein the antibody is administered systematicallyor locally.
 18. The method of claim 9, wherein the antibody is amonoclonal antibody.
 19. The method of claim 9, wherein the antibody isa human monoclonal antibody.
 20. The method of claim 9, wherein theantibody is a humanized antibody.